iUNIVERSITY OF MEDICINE AND FARMACY „VICTOR BABE ܇ ,” TIMI܇OARA FACULTY OF MEDICINE Departament of Functional Sciences Chair of Pathophysiology… [602211]

iUNIVERSITY OF MEDICINE AND FARMACY „VICTOR BABE ܇ ,”
TIMI܇OARA

FACULTY OF MEDICINE
Departament of Functional Sciences
Chair of Pathophysiology

DAVID-BENJAMIN EHRHARD

THESIS

MODULATION OF MITOCHONDRIAL FUNCTION BY NOVEL
ATP-DEPENDENT POTASSIUM CHANNEL OPENERS

Scientific Coordinators
Prof. Dr. Danina Muntean
Lect. Dr. Oana Duicu

T i m i ܈ o a r a
2 0 1 5

iiLIST OF ABBREVIATIONS ……………………………………………………………………………. iii 
PART I. GENERAL NOTIONS …………………………………………………………………………. 1 
1. Introduction ………………………………………………………………………………………………………………. 1 
2. ATP-sensitive potassium channels ………………………………………………………………………………… 2 
3. MitoK ATP channels ……………………………………………………………………………………………………… 3 
4. Openers and inhibitors of mitochondrial potassium channels ……………………………………………… 4 
4.1. Openers of mitoK ATP ……………………………………………………………………………………………… 4 
4.2. Inhibitors of mitoK ATP ……………………………………………………………………………………………. 5 
5. Consequences of mitoK ATP opening in cardiac mitochondria ……………………………………………… 5 
5.1. Mitochondrial swelling and matrix alkalinisation ………………………………………………………. 5 
5.2. Mitochondrial membrane potential and Ca2+ uptake …………………………………………………… 6 
5.3. Mitochondrial electron transport chain (ETC) inhibition …………………………………………….. 6 
5.4. Mitochondrial uncoupling and mild uncoupling …………………………………………………………. 7 
5.5. MitoK ATP-openers or protonophores? ………………………………………………………………………. 9 
5.6. Modulation of mitoK ATP channels and ROS production ……………………………………………… 10 
PART II. PERSONAL CONTRIBUTIONS ………………………………………………………..12 
1. Introduction and objectives ………………………………………………………………………………………… 12 
2. Materials and methods ………………………………………………………………………………………………. 13 
2.1. High resolution respirometry studies ……………………………………………………………………… 13 
2.2. Mitochondrial H 2O2 production assessment …………………………………………………………….. 18 
2.3. In vitro sensitivity to Ca2+-induced mPTP opening …………………………………………………… 19 
2.4. Chemicals …………………………………………………………………………………………………………. 21 
2.5. Statistical analysis ………………………………………………………………………………………………. 21 
3. Results ……………………………………………………………………………………………………………………. 22 
3.1. High resolution respirometry studies ……………………………………………………………………… 22 
3.2. Mitochondrial H 2O2 production …………………………………………………………………………….. 28 
3.3. In vitro sensitivity to Ca2+-induced mPTP opening …………………………………………………… 29 
4. Discussion ………………………………………………………………………………………………………………. 31 
PART III. CONCLUSIONS ………………………………………………………………………………34 
ORIGINAL FINDINGS AND FUTURE DIRECTIONS ………………………………………35 
ACKNOWLEDGEMENTS ……………………………………………………………………………….36 
REFERENCES…………………………………………………………………………………………………37 
ANNEX ……………………………………………………………………………………………………………43 

iiiLIST OF ABBREVIATIONS
ADP, D adenosinediphosphate
Ama antimycin A
ANOVA analysisofvariance
ATP adenosinetriphosphate
BSA bovine serumalbumin
c cytochrome c
C I complex I
C II complex II
CHD Coronary heart disease
DTNB 5,5’-dithio-bis (2-nitrobenzoic acid)
DTT Dithiothreitol
EDTA ethylenediaminetetraaceticacid
EGTA ethyleneglycoltetraaceticacid
ETC electrontransportchain
EtOH ethanol
F, FCCP carbonylcyanide p-(trifluoro-methoxy) phenyl-hydrazone
FBS fetal bovine serum
G glutamate
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
IPC ischemicpreconditioning
IpostC ischemicpostconditioning
I/R ischemia/reperfusion
M Malate
MES 2-(N-morpholino)ethanesulfonicacid
Omy oligomycin
OXPHOS oxidative phopsphorylation
PBS phosphate-bufferedsaline
RCR Respiratory control ratio
ROS Radical oxygen species
ROX Residual Oxygen consumption
Rot rotenone
S succinate
S.E.M Standard error of mean
SUIT substrate-uncoupler-inhibitor titration
TES 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid

1 PART I. GENERAL NOTIONS
1. Introduction
According to the World Health Organization, cardiovascular disease represents the number
one cause of death globally [1]. Particularly in developing countries, coronary artery
disease is the major cause of mortality due to myocardial infarction, and of morbidity due
to heart failure worldwide, respectively. Indeed, each year, acute myocardial infarction
(AMI) is responsible for the death of millions of humans worldwide and represents the first
cause of chronic heart failure. It has been predicted that, by the year 2020, AMI will be the
first cause of death in the world [2]. Timely coronary reperfusion by either thrombolysis or
primary coronary artery angioplasty has become the established routine therapy, which
effectively decreases infarct size and reduces mortality.
Since the 1960s, thrombolytic therapy began to advance and improve. This idea of therapy
w o r k s by ly si s of a t h r o m bo t i c oc c l usi on in th e c or on a ry ve s se ls of a p a ti en t t o a c hi ev e
reperfusion of the infarcted tissue. The latter will reduce infarct size, preserve left
ventricular function, and therefore will improve survival substantially [3]. In the modern
treatment of acute myocardial infarction, these interventions have become so immensely
important, that doctors denominated our times as the “reperfusion era” [4]. Knowing these
positive aspects of modern myocardial infarction therapy, one has to be aware of the
negative effects as well. With the rise of the pharmacological reperfusion therapy, another
phenomenon came to light: the reperfusion injury.
The distinctive pathophysiological changes associated with revascularization, collectively
called “reperfusion injury”, comprise: (i) reversible , sublethal events such as reperfusion-
induced arrhythmias and myocardial stunning (prolonged but fully reversible contractile
dysfunction) and (ii) irreversible , namely the induction of lethal reperfusion injury in
tissue that was potentially viable in the moments before reperfusion. Although the
existence of the latter was a matter of debate [5], further evidence supported the facts that
irreversible reperfusion injury through necrosis and apoptosis exists, and that the first
minutes of reperfusion represent a window of opportunity for the delivery of adjunctive
therapies [6, 7]. Accordingly, despite the unequivocal beneficial effects in stopping the
progression of irreversible damage, it is nowadays accepted that: (i) reperfusion per se is
considered a double-edged sword [8] as it can induce severe myocardial lesions itself, known
as lethal reperfusion injury, which paradoxically alleviates the beneficial effects of

2 revascularization [9], and (ii) there is currently no clinically available therapeutic
intervention able to further reduce infarct size in association with the revascularization
procedures [10]. Accordingly, cardioprotection at reperfusion, defined as the totality of
mechanical and pharmacological interventions aimed at reducing cell death during
postischemic reperfusion, nowadays represents the focus of considerable research for both
understanding the underlying mechanisms and translating the experimental findings into
clinical therapy [11-13]. Over the past decade, mitochondria, the well-known “power
plants” of the cell, producing the ATP to support the normal cell function, have emerged as
central regulators of cell death in a variety of disease states, including the ones associated
with ischemia-reperfusion injury [14] as well as in cardioprotection against it [15]. Several
research groups, including the one at the Department of Pathophysiology of our University
that hosted the experiments I will further describe, have systematically studied the
properties of different pharmacological compounds that might provide cardioprotection in
the setting of ischemia/reperfusion injury.
In this dissertation I will present the potential effects of two novel therapeutic agents,
which modulate the function of the ATP-dependent potassium channel. We worked on
experiments concerning this often investigated mitochondrial structure, and I will present
the corresponding results.
2. ATP-sensitive potassium channels
ATP-sensitive potassium channels (K ATP ) are types of potassium channels which have a
large distribution throughout the body and are present in a vast number of tissues including
muscle, pancreatic beta cells, and the brain [16]. They function as potassium uniporters,
allowing ion intake into the cellular matrix. In physiological conditions, K ATP channels are
closed by the direct binding of ATP, whereas the activation of K ATP channels occur due to
a reduced ATP/MgADP ratio that appears in energetic depletion periods such as ischemia.
Other factors including O 2, reducing equivalents/free radicals, pCO 2/pH and CO can
regulate the activity of K ATP channels as well [17]. K ATP channels were discovered first by
Noma in 1983 in the cardiac sarcolemma (sarcK ATP ) [18]. Later, in 1991, Inoue et al.
described a second type of K ATP channels located in the internal mitochondrial membrane
of rat hepatocytes, which were denominated mitoK ATP [ 1 9 ] . A l t h o u g h t h e r e w a s
remarkable progress in the last 20 years regarding the role, structure and mechanism of
action of K ATP channels, several questions still remain unanswered.

3 3. MitoK ATP channels
In modern cell biology, mitochondria are known to be the powerhouses of the cell. At the
same time they are involved in major processes such as apoptosis and cardioprotection.
After the discovery of mitoK ATP , the major goal was to determine their role in the
protection of cardiomyocytes. In 1997, Garlid et al. suggested for the first time that
mitoK ATP , rather than sarcK ATP , have the main role in cardioprotection. They tested the
effects of diazoxide on isolated cardiac mitochondria and determined that diazoxide was
1000 times more selective for mitoK ATP than for sarcK ATP . Also it was shown to be
cardioprotective in a model of I/R; these protective properties were abolished by 5-
hydroxydecanoic acid (5-HD), a selective mitoK ATP inhibitor [20]. The opening of the
mitoK ATP channels has been associated with beneficial effects for the cell, such as
increased ATP production and preservation of the latter during ischemia, increased
functional recovery at reperfusion [20], preservation of mitochondrial structure [21] and
infarct size reduction [22]. As it was shown by many studies, there are various mechanisms
through which the opening of mitoK ATP channels can induce cardioprotection. In
1998,Holmuhamedov et al. demonstrated that the functions of isolated rat heart
mitochondria can be modulated by opening of the mitoK ATP channels with
pharmacological mitoK ATP channel openers. They showed a consecutive membrane
depolarization, decreased Ca2+ uptake, an increase in matrix volume (referred to as
mitochondrial swelling), decrease in the rate of ATP synthesis, and the release of
mitochondrial proteins [23].
The opening of mitoK ATP channels is thought to elicit three direct effects on mitochondrial
physiology: (i) the increase of matrix volume, (ii) matrix alkalinisation, and (iii)
respiratory stimulation (uncoupling), which subsequently seem to have an effect on ADP
preservation during ischemia, prevention of Ca2+ overload, increased energy transfer
between mitochondria and cellular ATPases, inhibition of the mitochondrial permeability
transition pore (mPTP), and the prevention of apoptosis. All these effects strongly suggest
the important role of opening of the mitoK ATP channels in cardioprotection [24]. Other
protective mechanisms were proposed by Kopustinskiene et al.: inhibition of ATP
synthesis in the course of ischemia with a consecutive decrease of mitochondrial Ca2+
overload, mitochondrial swelling, modulation of ROS production during ischemia and
reperfusion, inhibition of mitochondrial respiration with a successive activation of ROS
signaling pathways, and a mild uncoupling effect [25]. Other authors suggest that the

4 mitoK ATP channel opening has little effect on mitochondrial respiration, and therefore on
ǻȌm and ǻpH , but th at the m ain eff ec ts are on the matrix an d the in te rmembrane space
volume [20].
4. Openers and inhibitors of mitochondrial potassium channels
Researchers, until today, have troubles finding out the molecular structure of mitoK ATP
channels. Despite of that, their functions and implications in cardioprotection have been
characterized by pharmacological means: compounds that can open or inhibit mitoK ATP
channels.
4.1. Openers of mitoK ATP
One problematic aspect was the lack of selectivity of compounds on the mitoK ATP
channels. The first tested compound was cromakalim, a benzopyran derivate which proved
this exact lack of selectivity [26]. Unselective agents such as cromakalim, izoflurane,
sildenafil, pinacidil, minoxidil, levosimendan, aprikalim, P-1060 and EMD 60480 open
both sarcK ATP and mitoK ATP channels. The first discovered selective mitoK ATP channel
opener was BMS 191095 [27]. It was proven to induce cytoprotection and inhibition of
Ca2+ influx into the mitochondrion in C2C12 myoblasts injured by treatment with H 2O2.
Long-term use can achieve a decrease of basal ROS production, an improvement in ATP
homeostasis [28], and a lack of action potential shortening activity [27]. Despite its
outstanding efficiency, BMS 191095 cannot be used in clinical practice because of its
neuronal toxicity [29]. Other discovered selective mitoK ATP openers were nicorandil,
which is a pyridinecarboxamide, and diazoxide, which is the prototype for
benzothiadiazines. The latter is most commonly used in animal model experiments,
although it has been reported that the opening of the mitoK ATP channels in its case is not
with absolute selectivity. It is shown to be 2000 times more selective on mitoK ATP than on
sarcK ATP channels [20], and also it was recently reported that diazoxide presents
cardioprotective mechanisms which are mitoK ATP independent, one of which being the
inhibition of succinate dehydrogenase [30].

5 4.2. Inhibitors of mitoK ATP
To further test the functions of the mitoK ATP channels, a lot of interest was shown for the
inhibitors of mitoK ATP channels as well. They are able to abolish the cardioprotective
effects, which are previously induced by mitoK ATP openers or by preconditioning of the
cells. Mostly used is 5-HD, which is selective for mitoK ATP channels.
5. Consequences of mitoK ATP opening in cardiac mitochondria
5.1. Mitochondrial swelling and matrix alkalinisation
It is known that the mitochondrial matrix will contract in pathological conditions. This
contraction is leading to an increase in distance between the outer and the inner
mitochondrial membrane. That contraction was shown to be counteracted by mitoK ATP
channel openers through matrix swelling [31]. The swelling is induced by K+ influx
through the mitoK ATP channels, which is accompanied by osmotically obligated water and
Pi entering the mitochondria. Laclau et al. were the first to prove that the mitoK ATP channel
opening will lead to an increase of the steady-state volume of 15-20% [32]. This effect was
also proven to be inhibited by the administration of 5-HD [33]. Interestingly, ATP will
inhibit the increase in matrix volume, but this action is inverted by the administration of
diazoxide[24]. Kowaltowski et al. made similar observations, and also showed that
mitoK ATP opening by diazoxide can induce, even in absence of respiration, a functional
response of the mitochondria.
As it was shown in several experiments, mitoK ATP opening during I/R can substantially
improve the energetic metabolism, and this can be counteracted by mitoK ATP inhibitors
l i k e 5 – H D [ 3 4 ] . A s a m a t t e r o f f a c t , A T P s y n t h e s i z e d b y o x i d a t i v e p h o s p h o r y l a t i o n i s
t r a n s p o r t e d i n t o t h e c y t o s o l b y s e v e r a l d i s t i n c t c o m p l e x e s , w h i c h a r e l o c a t e d i n t h e
mitochondrial membranes: ATP/ADP translocator (ANT), mitochondrial creatine kinase
(Mi-CK), and the voltage-dependent anion channel (VDAC). In stress conditions, as stated
before, the mitochondrial intermembrane space will increase in size due to matrix
contraction. The ATP translocation function will decrease in those conditions, due to the
reduction of the contact between the complexes. Because matrix swelling and a subsequent
decrease of mitochondrial intermembrane space occurs after the administration of
mitoK ATP channel openers, we can expect to prevent those effects by inducing a better
f un c ti on al c o upl i ng f or th o s e A T P t ra nsl oc a ti on c om p l e x es . I n f a c t , i t w a s sh o wn th a t a

6 mitoK ATP channel opening with diazoxide in isolated mitochondria induced a stronger
contact between ANT, Mi-CK, and VDAC, and therefore can be responsible for the
preservation of structure and function of the inner mitochondrial space, the low
permeability of the outer membrane to nucleotides, and so substantially contributes to the
maintenance of energy transfer during reperfusion after ischemia [32, 35].
Matrix alkalinisation is a phenomenon which is strongly associated with mitoK ATP channel
opening and consecutive K+ influx. Overall transport in mitochondria must be
electroneutral. The opening of mitoK ATP channels will generate a K+ influx, which will be
balanced by the electron transport chain (ETC) by means of H+ efflux, which will further
induce an increase of the pH in the matrix. The Pi transporter tries unsuccessfully to
compensate this inequality by an electroneutral uptake of Pi, and consequently the matrix
alkalinisation will occur[36]. The latter will also determine a mild uncoupling of Complex
I (CI), and an increased ROS production [37].
5.2. Mitochondrial membrane potential and Ca2+ uptake
It has been shown that metabolic stress situations will lead to an increased cytosolic Ca2+,
which in turn will interfere with mitochondrial homeostasis, and can lead to cellular
energetic failure and cell death. Therefore, another possibility to elicit cardioprotection is
the prevention of mitochondrial Ca2+ overload. Fortunately , this was shown by mitoK ATP
channel opening. Due to the mitochondrial depolarization after mitoK ATP channel opening,
a reduced mitochondrial Ca2+ loading in isolated mitochondria as well as in intact
cardiomyocytes was proven. This action was K+ dependent and sensitive to inhibitors of
mitoK ATP ch an n el s [ 3 8] . D ue t o t h e f a c t th a t t he Ca2+uniporter is potential dependent, it
was suggested that the mitoK ATP channel opening, and the successive K+ influx, determine
a membrane depolarization which will inhibit the Ca2+ uniporter, and prevent excessive
Ca2+ overload [21].
5.3. Mitochondrial electron transport chain (ETC) inhibition
T o f u l l y u n d e r s t a n d t h i s n e x t c h a p t e r , a s h o r t s u m m a r y o f t h e E T C s a c t i o n s i s f u r t h e r
presented. According to Peter Mitchell’s chemiosmotic theory (1966), the oxidation of
substrates at the level of complex I (CI-NADH dehydrogenase) and complex II (CII-
succinate dehydrogenase) will produce electrons that are transported along the complexes
of the ETC to complex IV (C IV: cytochrome c oxidase), where intracellular oxygen

7 undergoes a tetravalent reduction to water. Consecutively with the electron passing
t h r o u g h , t h e e n e r g y p r o d u c e d i s u s e d b y t h r e e c o m p l e x e s C I , C I I I ( c y t o c h r o m e c
reductase), and CIV to pump protons from the mitochondrial matrix into the
intermembrane space, therefore generating a proton-motive force ( ǻp) comprising other
two components, the mitochondrial membrane potential ( ǻȌm) and the pH gradient
ǻpHm) . Th e ǻp g en e r a t e d i s u se d t o m a in ta i n t he i on h ome o s t as i s , t o a l l ow t h e pr o t e in
import for mitochondrial biogenesis and for the proton translocation through the F0 part of
ATP synthase from the intermembrane space back in the mitochondrial matrix, a process
that is coupled with the phosphorylation of ADP in ATP [15, 39].
Two different groups of researchers proposed that the association between cardioprotection
and mitoK ATP channel opening are mediated by the inhibition of the ETC at different
complex levels, namely CI [40] or CII [41]. Numerous studies showed that mitoK ATP
channel opening with successive modulation of mitochondrial respiratory function can
induce cardioprotection through uncoupling and reversible inhibition of the ETC [20, 30,
40]. It was also hypothesized that the cytoprotective properties of diazoxide are mediated
solely by the inhibition of ETC, independently of mitoK ATP channel opening [42]. In an
experiment carried out by blockage of ETC with amobarbital (reversible blockage of ETC
at CI), Chen et al. observed that CI, CIII, and CIV were protected during ischemia,
cytochrome c release was inhibited, the mitochondrial membrane integrity and the
respiratory function were preserved [43], and the ROS production following reperfusion
decreased [44]. If there is no ischemic damage of the electron transport chain, the
reperfusion of myocardium can reduce the infarct size, improve contractile function
recovery, and decrease mitochondrial Ca2+ overload [45]. So as it seems, mitochondrial
dysfunction at mild degrees elicited by modulating respiration is rather protective than
deleterious at reperfusion [46]. Thus, an important strategy in cardioprotection appears to
be the reduction of ATP consumption during ischemia [14]. The latter goes along with the
beneficial effects of ischemic preconditioning [47] and administration of diazoxide [35].
5.4. Mitochondrial uncoupling and mild uncoupling
In 1948, the term “uncoupling” was introduced for the first time by Loomis and Lipmann
[48]. As stated above, the ETC will pump protons(H+) from the mitochondrial matrix into
the mitochondrial intermembrane space, which are reintroduced into the matrix through the
ATP synthase, producing ATP. The term “uncoupling” was introduced to denominate any

8 mechanism which can produce a proton passage from the mitochondrial intermembrane
space back into the mitochondrial matrix, which is not mediated by the ATP
synthase/complex V [49]. Thus, pharmacological uncoupling agents can interrupt the link
between substrate oxidation and the phosphorylation of ADP to ATP. Therefore, when
uncoupling, a collapse of H+ chemical potential will be induced [50]. Two types of
uncoupling were described [51]. The first type, called extrinsic uncoupling of oxidative
phosphorylation (oxphos), will increase the proton/cation permeability of the membranes,
and will induce a decrease or even a collapse of the proton motive force ǻp. The intrinsic
uncoupling (slipping) of oxidative phosphorylation will result in a discrepancy between
chemical turnover and transport activity, and therefore leading to decreased
phosphate/oxygen ratios (P/O ratio).
A s s ta te d by T e r a d a i n 1 99 0, m os t of th e un c o up l e r s of oxi da ti v e ph os ph o ry l a ti on w e re
supposed to be hydrophobic weak acids which must have protonophoric actions, meaning
that they are able to transport H+ over an H+- impermeable membrane, and therefore being
able to induce a collapse of the H+ chemical potential [50]. Actually, any physical force or
pharmacological compound which is able to dismantle the mitochondrial membrane
potential ( ǻȌm) and/or the pH gradient ( ǻpHm)can elicit uncoupling.
The mitochondrial respiratory rate is a value that can be precisely measured nowadays.
When inducing mitochondrial uncoupling, one will see certain changes of the
mitochondrial respiratory rate. The mitochondrial respiration is measured in the presence
of different substrates (glutamate/malate; pyruvate/malate; succinate). In basal conditions,
the respiratory rate is low, and is called “state 2”. To induce the increase of respiratory
rates and to continue to “state 3”, exogenous ADP is added. The mitochondria will return
to state 2 when all the available ADP is phosphorylated into ATP [50]. State 3 will
transition into state 4 when adding compounds which will inhibit the phosphoryl transfer
(e.g., oligomycin- inhibits H+ passing through F0F1-ATPase). Then, the addition of an
uncoupling agent will induce the release of this inhibited respiration, and will also increase
state 2 in a dose-dependent way [50]. The respiration can then be inhibited by adding
compounds like antimycin or KCN (potassium cyanide). After the respiration was
inhibited, uncoupling agents are not able to stimulate the respiration again [50]. It was
stated that an uncoupling compound has to possess a high hydrophobicity and electron-
withdrawing properties, and that without those properties these compounds would not be
able to accomplish uncoupling [50]. In other words, adding an uncoupling agent will

9 determine an increase in the respiratory rates of state 2 and state 4, and a significant
increase in O 2 consumption [50, 52-54].
Uncoupling and cardioprotection were first associated when studies showed that an
activation of survival pathways by ischemic preconditioning can lead to antiischemic
myocardial protection [55]. It was shown by Morota et al. in 2013, that extensive
uncoupling of mitochondria can even exacerbate the ischemic injury, but on the other
hand, if administered in small concentrations and therefore inducing mild uncoupling, can
elicit cardioprotection [52, 56]. Brennan demonstrated in 2006, that the pre-treatment of
I / R h e a r t s wi th l ow d os e s of F CCP ( 1 00 ȝM w a s c on si de r e d th e op ti m al c on cen tr a ti on )
which induces partial uncoupling, improved the post ischemic functional recovery [52].
With moderate uncoupling, one can obtain a decrease of the Ca2+ overload and a decreased
production of ROS, which both alleviate the ischemic injury [30]. Liu et al. described in
1999 that the administration of diazoxide and pinacidil, which induce major uncoupling,
decreased the mitochondrial Ca2+ overload and therefore promoted cardioprotection [57].
5.5. MitoK ATP -openers or protonophores?
As stated above, it was demonstrated that the opening of mitoK ATP channels by diazoxide
and pinacidil have cardioprotective properties by acting as uncoupling protonophores and
promoting the transmembranary proton translocation [30]. They showed their uncoupling
action by accelerating state 4 respiration, reduction of the mitochondrial membrane
potential, and the decrease of ATP production in the mitochondria. For some reason, this
uncoupling was insensitive to 5-HDand was maintained in nominally K+-free medium [30].
Experiments suggested that diazoxide and pinacidil facilitate H+ transport and generation
of H+-selective currents across membranes, therefore meeting all the criteria to be called
protonophores. This suggests that those properties can represent a supplementary
component of mitoK ATP action [30]. In 2001, Kowaltowsky et al. induced a decrease of the
mitochondrial membrane potential ( ǻȌm) and Ca2+ uptake by administering high doses of
diazoxide and pinacidil, and demonstrated that this happened without any relation to
mitoK ATP opening, by incubating the mitochondria without ATP and Mg2+, and therefore
leaving the mitoK ATP channels in an open state, rendering them insensitive to any action of
mitoK ATP openers [58]. This therefore suggests another mechanism of action, without
mitoK ATP opening. The final conclusion of those experiments was, that the effects of
diazoxide and pinacidil can be accredited to uncoupling via an intrinsic protonophoric

10 activity and respiratory inhibition at high dosages(> 100 ȝM and > 50 ȝM, respectively),
rather than effects on the influx through mitoK ATP channels [58]. Similar evidence was
found by Hanley et al. in 2002 and Dröse et al. in 2006 [59, 60].
5.6. Modulation of mitoK ATP channels and ROS production
T h e r e a r e s o m e a p p r o a c h e s w h i c h c a n b e u s e d t o e l i c i t c e l l u l a r p r o t e c t i o n , s u c h a s t h e
modulation of: ATP production, mitochondrial respiration, ion transport, redox state, free
radical release, and cytochrome c release.
Mitochondria seem to be the major players in ROS production. An inequality between
ROS formation and defenses against the produced antioxidants is represented by oxidative
stress to the cell. The first ROS to be produced is superoxide anion (O 2-), although it will
be rapidly transformed to hydrogen peroxide (H 2O2) by superoxide dismutase (SOD) or by
a spontaneous dismutation [61]. H 2O2 can be entirely reduced to water or partially reduced
to hydroxyl radical (OH-), which is one of the strongest oxidants occurring in nature,
which in turn may be re-reduced by O 2- [62].
ROS generation in mitochondria was shown to be regulated by the proton motive force
ǻȌm and ǻpH) [63], which will inhibit the proton pumps of the respiratory chain at high
levels [64]. There are some main ROS production sites proposed by different authors. In
r e c e n t s t u di e s , c o m pl ex I an d c o m pl ex I I I w e r e m o s t of t e n n a m e d a s i m p or t a n t si t e s f or
ROS production [65]. Each site will react differently to changes in the proton motive force.
If one inhibits the regulatory sites, an increase of ROS production will follow [61]. It was
also proposed, that increased mitochondrial proton conductance, thus uncoupling, leads to
the oxidation of ubiquinone and to a decreased ROS production, which in turn leads to
reduced oxidative stress and preventing the cell from ageing [66]. ROS production seems
to be decreased by high respiratory rates. A high respiratory rate will increase the oxygen
consumption, which in turn leads to lower oxygen tensions and lesser reduction of oxygen
to reactive oxygen species at the electron transport chain. High respiratory rates also
increase the NAD+ availability, which decreases ROS release by pyruvate and Į-
ketoglutarate dehydrogenases [67].
As the reduction of the membrane potential is associated with limiting the oxidative
damage due to the decrease of ROS production[68], membrane hyperpolarization is
associated with an increased ROS production and even with apoptotic events. To reduce

11 ROS production by a number of 90%, one has to increase the basal respiration (e.g. state 2)
by 200%. This means that the uncoupling effect should be elicited in a manner that will
still allow ATP synthesis [69].

Figure 1. Modified after Brondani et al, Arq Bras Endocrinol Metab , 2015

12 PART II. PERSONAL CONTRIBUTIONS
1. Introduction and objectives
S t a r t i n g w i t h th e d i s c ov e ry o f t h e K ATP channels, there has been ever growing research
f o c u s e d o n p h a r m a c ol o g i c a l a g e n t s , w h i c h w o ul d h a v e t h e a b i l i ty t o m o d u l a t e t h e K ATP
channels, both in vitro and in vivo. Several K ATP channel openers as well as inhibitors with
diverse chemical structures have been characterized, e.g. Benzopyrans (BMS 191095),
Benzothiadiazines (diazoxide), and Cyanoguanidines (pinacidil). This increased interest in
modulators of K ATP channels grew bigger with the discovery of the connection between
KATP channels and cardioprotection against ischemia-reperfusion injury [70]. It is widely
accepted nowadays that K ATP channels are responsible for the coupling of cellular
metabolism with the membrane activity/electrical activity in diverse types of cells,
including myocardial cells [71]. It was shown, that the activation of K ATP channels in
periods of metabolic inhibition can induce a notable shortening of the action potential and
a sequenti al reducti on of contrac tili ty . Thi s process can l ea d to the con serv ation of ATP
and the contractile function of the cell, and therefore to cardioprotection [72].
The uncoupling effect represents any mechanism that can produce the proton (H+) passage
from the inner mitochondrial space back into the matrix, but not through ATP synthase
[49]. An extensive uncoupling of mitochondria determines negative effects on normal
mitochondrial function (by decreasing the intracellular ATP production, contraction force,
and duration of action potential), therefore exacerbating the ischemic injury (42).
However, small concentrations of uncoupling agents can induce cardioprotection by "mild
uncoupling" (38). "Mild uncoupling" has been suggested to be associated with reduced
sensitivity of mitochondria to opening of the mitochondrial permeability transition pore
(mPTP), reactive oxygen species (ROS) generation, Ca2+ uptake, and mitochondrial
membrane potential (38-40). Considering this, an apparently counterintuitive concept has
emerged, that a mild degree of mitochondrial dysfunction elicited by modulating
respiration is rather protective than deleterious at reperfusion [46]. However, the lack of
selective mitoK ATP agents still represents a problem in demonstrating cardioprotection
conferred by these agents [73].
The aim of the present study was to characterize the effects of two novel synthetic
benzopyranyl analogues, designed as selective mitoK ATP openers, on mitochondrial
function.

13 The specific objectives were as follows:
1. To learn the isolation technique of rat heart mitochondria.
2. To assess the effects of two novel synthetic pharmacological openers of mitoK ATP ,
KL-1492 and KL-1507, on: i) mitochondrial respiratory function, ii) ROS
production, and iii) in vitro sensitivity to Ca2+-induced mPTP opening.
2. Materials and methods
All the experimental procedures used in this study were conducted in accordance with the
Directive 2010/63/EU and the corresponding Romanian law nr. 43/May 2014 regarding the
protection of animals used for scientific purposes, respectively. The experimental protocol
was approved by the Committee for Research Ethics of “Victor Babes” University for
Medicine and Pharmacy of Timi úoara, Romania.
2.1. High resolution respirometry studies
The isolation of the rat heart mitochondria (Fig. 2) was performed by a procedure
previously described by O. Duicu in 2013 [74]. The animals were first anesthetized with an
intraperitoneal injection of ketamine (Vetased, 30mg/kg) and xylazine (Xylazin, 10mg/kg).
The hearts were removed rapidly by the following method: the rat was pinned on a particle
board in supine position, and abdominal skin and fur was prepped away with tweezers and
a s ci ss or . T h e n , th e a bdo mi n a l c avi ty w a s op ene d. T h e th or a c i c ca v i ty w a s a c c es s e d by
opening the rib cage laterally and cutting the diaphragm from below (Fig. 1). Hearts were
removed by cutting the connections to the big vessels, and immersed into ice cold isolation
m e d i u m m a d e f r o m 1 0 0 m M s uc r o s e , 5 0 m M p o t a s s i u m c h l o r i d e , 2 0 m M T E S , 1 m M
EDTA and 0.2% fatty acid free BSA (bovine serum albumin) with a pH of 7.2 and 4șC.
Then the atria were clipped off and discarded. The ventricular tissue was minced and
t r e a t e d w i t h n a g a r s e ( 5 m g / g o f w e t c a r d i a c t i s s u e ) , w h i c h i s a p r o t e a s e t o p r o m o t e t h e
breakdown of the cellular structure. Then the tissue was homogenized in the isolation
medium, using a Polytron homogenizer with a Teflon tappet. The obtained homogenate
was centrifuged (Rotina 38R centrifuge) by differential centrifugations at 4°C (Fig. 2, D).
The first centrifugation was performed for 10 minutes at 8500 G and the resulting pellet
was again suspended in the incubation medium and manually homogenized, and
centrifuged for 10 minutes at 800 G.

14

Figure 1. Rat heart removal.
To obtain a higher purity of mitochondria, the resulting supernatant was again centrifuged
for 10 minutes at 8500 G.The final pellet of mitochondria was resuspended in a glass
homogenizer, kept on ice, and used within four hours in respiratory rates measurements.
The mitochondrial protein concentration was further evaluated, according to the Biuret
method [75].
Figure 2. Steps in mitochondria isolation. (A): Immersion of the heart in ice-
cold isolation medium; (B): After discarding the atria, both ventricles are very
well minced with thin scissors ; (C): Mechanical homogenization of the sample
in a Polytron homogenizer with a Teflon pestle; (D):Centrifugation.
To evaluate the respiration of the mitochondria, the OROBOROS® Oxygraph-2k
(Oroboros instruments, Austria) with DatLab software, was further used (Fig. 4). It’s the
only instrument world-wide which is able to perform “high-resolution respirometry”
(HRR). The oxygraph is measuring the oxygen concentration and consumption in the

15
incubation medium over time, while one performs various titrations which are presented in
a respirometric protocol [76].

Figure 3. Steps of differential centrifugations of rat heart mitochondria.
The oxygen consumption was measured at 37șC with the Oxygraph-2k. A standard oxygen
air calibration must be conducted before each respirometry measurements [77]. For the
latter, 2.5 ml of MIRO6 were introduced in both chambers, and the stoppers were inserted
slowly to the volume-calibration position (Fig. 4). Then the stoppers were lifted to leave a
small gas bubble above the liquid phase for final air equilibration. Stability of the
polarographic oxygen sensors (POS) signal (0±1pmol*s-1*ml-1) was normally reached
within one hour [77]. When the calibration was finished, the stoppers were slowly pushed
down into the chambers and the excess media was siphoned off. Afterwards, the
experiment was conducted according to a SUIT (substrates-uncouplers-inhibitors-titration)
protocol previously described [74]. The mitochondria (0.1 mg protein/ml) were incubated
i n 2 m l o f i n c u b a t i o n m e d i u m , M I R O 6 [ 7 8 ] c o n t a i n i n g 0 . 5 m M E G T A , 3 m M
MgCl 2.6H2O, 60 mM K-l ac tobi on ate, 20 mM ta urine, 10 mM KH 2PO4, 20 mM H EPE S,

16
110 mM sucrose, 1 g/l BSA, essentially fatty acid free + 280 U/ml catalase lyophilized
powder, 2000-5000 units/mg protein (pH 7.1, 37șC).

Figure 4. The OROBOROS Oxygraph-2k: air calibration position
A standard oxygen air calibration was performed before each respirometric measurement
[77]. For the latter, 2.5 ml of MIRO6 were introduced in both chambers, and the stoppers
were slowly inserted to the volume-calibration position, to leave a small gas bubble above
the liquid phase for final air equilibration. Stability of the polarographic oxygen sensors
(POS) signal (0±1pmol*s-1*ml-1) was normally reached within one hour [77] and R1
mark was selected for air calibration and inserted in the air calibration window (Fig. 5).
When the calibration was finished, the stoppers were slowly pushed down into the
chambers and the excess media was siphoned off. Afterwards, one can continue with the
SUIT protocol.

17

Figure 5. Stabilization of the polarographic oxygen sensors signal: maximum 60 min calibration, at 37°C.
The bottom graph (redline) shows the block temperature; the green line shows the relative power at which the
Peltier system regulates the block temperature. Thermal equilibrium is reached when both temperature and
Peltier power are constant. The top and middle graphs show the negative slope of the oxygen concentration
over time for the right and left chamber (red line), respectively, expressed as pmol·s-1·ml-1 on the right Y-
axis, with zero in the middle position. A slope of zero („O2 Slope uncorrected ெ) indicates a constant oxygen
signal over time. (A): R1 marker is inserted in the calibration window.

In our study, we wanted to evaluate the complex I-dependent respiration in chamber A,
and the complex II-dependent respiration in chamber B. Thereby, the SUIT protocol used
in our experiments consisted of the following steps:
1) The addition of 10mM glutamate and 2mM malate (CI substrates) into chamber A,
and 10mM succinate (CII substrate) and 0.5µM rotenone (CI inhibitor) into
chamber B: state 2 (basal respiration);

2) T h e a d d i t i o n o f 5 m M A D P : s t a t e 3 o r O X P H O S : t h e m a x i m a l o x i d a t i v e
phosphorylation capacity;

18 3) Addition of 10µM cytochrome c. During the isolation steps of the mitochondria, the
mitochondrial membrane integrity can be damaged. If that happens, cytochrome c
will be released, and will limit the mitochondrial respiration. One property of
cytochrome c is, that it cannot penetrate the outer mitochondrial membrane when
its intact. Thus, the absence of an increase of respiration rate after adding 5-32 ȝM
cytochrome c indicates a high quality preparation and a quality control measure of
mitochondrial membrane integrity [79];

4) Addition of oligomycin (2 µg/ml) to inhibit the F 0F1-ATP synthase: state 4 of
respiration;

5) The titration of a protonophore, in this case FCCP, in 0.5 µM steps, to obtain
uncoupled respiration and to evaluate the electron transport system (ETS) capacity.
Due to the uncoupling, the electrochemical proton potential across the inner
mitochondrial membrane will collapse, leading to a relief of electrochemical
pressure elicited on the proton pumps (CI, CIII, and CIV).

6) A d d i t i o n o f 2 . 5 µ M a n t i m y c i n A , t o i n h i b i t t h e r e s p i r a t i o n : t h e r e s i d u a l o x y g e n
consumption – ROX state.

Mitochondrial respiration was further corrected for oxygen flux due to instrumental
background and ROX [80].
The respiratory control ratio (RCR), as a classical parameter for the mitochondrial
qualitative control, indicating the coupling between oxygen consumption and oxidative
phosphorylation, was calculated as the ratio OXPHOS/ State 4.
2.2. Mitochondrial H 2O2 production assessment
The main sites for ROS production are the complexes I and III of the electron transport
chain. ROS are either generated from electron transfer after oxidation of the substrates of
complex I and II, or from the same complexes in a reversed order, namely from complex II
to complex I. Studies suggest that the most relevant ROS production site is complex I after
reverse electron flow [81]. O2- will dismutate either spontaneously or by the help of
superoxide dismutase (SOD) into hydrogen peroxide (H 2O2) which can diffuse through

19 biological membranes and is more stable. Therefore, the measurement of H 2O2 can be
used as an indicator for ROS production.
Mitochondrial H 2O2 production was measured using the Amplex Red (10 ȝM) fluorescent
marker, as previously described by Duicu et al. in 2013 [74]. Amplex red is a non-
fluorescent dye, which in the presence of horseradish peroxidase will be oxidized
enzymatically by H 2O2 and become fluorescent. The excitation wavelength was set to 530
nm and the emission wavelength to 590 nm. Mitochondria (0.25 mg protein/ml) were
i n c u b a t e d i n 2 m l i n c u b a t i o n b u f f e r ( 2 5 0 m l s u c r o s e , 1 m M E G T A , 1 m M E D T A , 2 0
mMTris/HCl and 1.5 mg/ml defatted BSA, pH 7.4) at 37șC, supplemented with CI-
dependent substrates: G (5 mM) and M (5 mM). The fluorescence signal was calibrated by
adding known amounts of H 2O2 to the incubation buffer in the presence of Amplex Red
and horseradish peroxidase. At the beginning of each measurement, the background
fluorescence was quantified, in the absence of mitochondria. Net fluorescence was then
calculated by measuring the fluorescence variation in function of time, minus background.
Results were expressed as pmol H 2O2/min/mg proteins.
2.3. In vitro sensitivity to Ca2+-induced mPTP opening
To assess the in vitro sensitivity to Ca2+-induced mPTP opening we measured the calcium
retention capacity (CRC) according to a technique previously described [74]. According to
I c h a s e t a l . ( 1 9 9 4 ) , w e d e f i n e d C R C a s t h e a m o u n t o f C a2+ requested to induce an
important Ca2+ release by isolated cardiac mitochondria [82]. In this way , CRC is used as
an indicator of the resistance of the mitochondrial permeability transition pore (mPTP) to
opening after matrix Ca2+ accumulation.
T h e s t r u c t u r e o f m P T P i s s t i l l u n c e r t a i n a f t e r m o r e t h a n 3 0 y e a r s o f i n t e n s i v e
investigations. Still, two molecules located in the internal mitochondrial membrane (IMM)
were accepted as key structural components of mPTP: phosphate intracellular carrier (PiC)
and adenine nucleotide translocase (ANT) [83] (Fig. 6). Cyclophiline D (Cyp-D), located
in the mitochondrial matrix, is considered rather a modulator, but not a structural
component of the mPTP (Basso et al., 2005). The IMM is impermeable to most
metabolites and ions in physiological condition, but in pathological conditions, (i.e.,
increased Ca2+ level in mitochondrial matrix, enhanced ROS production, high phosphate
level, and low adenine nucleotides) mPTP will be formed in the IMM [83] (Fig. 6).

20
Opening of mPTP allows a passive diffusion of molecules of < 1.5 kDa, disrupting thus the
permeability of the IMM. This leads to mitochondrial uncoupling and swelling and in the
end to mitochondrial energetic failure [83]. As a consequence, the reduced ATP amount is
not longer able to maintain the structure and the function of the cell, ionic homeostasis is
altered, leading to cell death, predominantly via necrosis [84].
Figure 6. The schematical presentation of the structural components of the
mitochondrial permeability transition pore and its role in cell death.
(Modified after Halestrap, 2009). The mitochondrial permeability transition
pore (mPTP) is a “channel” localized in the inner mitochondrial membrane
(IMM), and assembled by the phosphate carrier and adenine nucleotide
translocase (PiC and ANT, in the IMM), cyclophilin D (CypD, in the
mitochondrial matrix). In physiological conditions mPTP is closed, and
mitochondrial ATP synthesis is normal, maintaing thus the living cells. In
pathological conditions, such as mitochondrial calcium overload, oxidative
stress, adenine nucleotide depletion, inorganic phosphate accumulation, the
mPTP opens, allowing the entrance in the mitochondrial matrix of all solutes <
1,5 kDaltons, leading to mitochondrial swelling and altered ATP synthesis, and
eventually to cell death. mPTP opening may be inhibited by cyclosporine A
(CsA), low pH, or Mg2+. (Illustration realized thanks to Servier Medical Art and
reproduced by permission of [85].


21 Cyclophilin D (Cyp-D), a nuclear encoded mitochondrial isoform of cyclophilin family,
forms a complex with a target protein, inducing a conformational change that generates
formation of a channel [86]. When CyP-D binds Cyclosporine A (CsA), it is unable to bind
to the target protein anymore (Fig. 6). Mitochondria isolated from Cyp-D knockout mice,
present a lower sensitivity to Ca2+, and thus a delayed mPTP opening, insensitive to
cyclosporine [87].
We monitored in our experiments the change in extramitochondrial Ca2+ concentration
using Ca-green-5N (1 ȝM) as fluorescent dye (excitation-emission, 500 – 530 nm).
Measurement of calcium uptake by isolated mitochondria (0.25 mg protein/ml) was
performed at 37°C in 2 ml incubation buffer (150 mM sucrose, 50 mM KCl, 2 mM
KH2PO4, 5 mM succinic acid in 20 mM Tris/HCl, pH 7.4, at 37°C) using a Hitachi F-7000
spectrofluorometer. CaCl 2 pulses (20 nmol/pulse) were added every minute until we
observed a sudden increase of the extramitochondrial calcium concentration, signaling the
opening of the mPTP. In the end, CRC was calculated as the total amount of Ca2+ entering
mitochondria before Ca2+ release.
We also measured the CRC in the absence/presence of 1 ȝM of CsA, the classical inhibitor
of mPTP, which was used in our experiments as a "positive" control of our method [88].
2.4. Chemicals
The benzopyranyl analogues were synthesized by Lorand Kiss at the Institute of
Pharmaceutical Chemistry, Faculty of Pharmacy, University of Szeged, Hungary. Stock
s o l u t i o n s w e r e m a d e u p i n D M S O ( 6 0 m M ) a n d t h e n s e r i a l l y d i l u t e d t o g i v e t h e
investigated concentrations (50, 75, 100, 150 µM) for each compound. The final DMSO
concentration was constant (0.25%) throughout the experiments and did not influence
respiratory rates. Calcium Green-5N and Amplex Red were purchased from Invitrogen. All
the other chemicals were developed by Sigma-Aldrich.
2.5. Statistical analysis
Data were expressed as means ± SEM. Data analysis used one-way ANOVA followed by a
post-hoc Tukey's multiple comparison test (GraphPadPrism v. 5.0 Software, SUA). The
difference was considered statistically significant if p<0.05.

22
3. Results
3.1. High resolution respirometry studies
Our SUIT protocol design of rat heart mitochondrial respirometry measurements is
presented in Fig. 7 and Fig. 8.

Figure 7. Representative diagram of the SUIT protocol for CI-supported respiration. A: CTRL; B: KL
1492 150ȝM; C: KL 1492 150 ȝM.Oxygen concentration (cO 2) and O 2 flux (pmol O 2*s-1*ml-1) are
represented as a function of time.Additions are as follows: G – glutamate (10 mM), M – malate (0.2 mM), D
– ADP (5mM), Cyt C – cytochrome c (10 ȝM), Omy – oligomycine ( 2 ȝg/ml), F – FCCP (0.05 ȝM), Ama –
antimycin A (2.5 ȝM). Oxygen concentration (maximum 200 nmols/ml)was maintained by intermittent
H2O2 additions into the chamber (100 ȝM at each titration step).

23

Figure 8. Representative diagram of the SUIT protocol for CII-supported respiration.A: CTRL; B:
KL 1492 150 ȝM; C: KL 1492 150 ȝM.Additions are as follows: Rot – rotenone (0.5 ȝM),S – succinate (10
mM), D – ADP (5mM), Cyt C – cytochrome c (10 ȝM), Omy – oligomycine ( 2 ȝg/ml), F – FCCP (0.05 ȝM),
Ama – antimycin A (2.5 ȝM)

Two benzopyran derivatives, namely KL-1492 and KL-1507, in four concentrations (50,
75, 100 and 150 µM, respectively) were screened and a concentration-dependent
modulation of mitochondrial respiratory function was found (Fig. 9 and Fig. 10).

24

Figure 9. HRR studies for CI-supported respiration in the presence of KL-1492 . Results are expressed in
pmol/(s*ml). Values are means ± SEM. p<0.05

25

Figure 10. HRR studies for CI-supported respiration in the presence of KL-1507 . Results are expressed
in pmol/(s*ml). Values are means ± SEM. p<0.05

Accordingly, a concentration-dependent increase in respiratory states 2 and 4 (Fig. 9 and
10: A, B) was found to become significant when they were added in the highest
concentrations (150 and 100 ȝM). The maximum increase was found for KL-1492 for both
state 2 (by 184.4% for 100 ȝM and 336.4% for 150 ȝM, Fig. 9 A) and state 4 (by 74.8%
for 100 ȝM and by 140.8% for 150 ȝM, Fig. 9 B), respectively. Lower, yet significant
changes were found in the case of KL-1507, namely state 2 increased by 53.2% at 100 ȝM

26 and by 79.4% at 150 ȝM (Fig. 10 A) whereas state 4 was increased by 64.2% and 64.6%
vs. Ctrl (Fig. 10 B).
However, when applied in the maximal concentration (150 ȝM), both compounds induced
a significant decrease in OXPHOS (State 3, ADP-stimulated respiration) vs. Ctrl for KL-
1 4 9 2 by 5 7 . 5 % , a n d f o r K L – 1 5 0 7 by 4 8 . 3 % , r e s pe c t i v e l y ( F i g . 9 , 1 0 : C ) . T h e s e e f f e c t s
(increase in state 4 together with the decrease in OXPHOS) for 150 ȝM ended up (as
expected) in a significant reduction of the respiratory index (OXPHOS/ State 4) vs. Ctrl for
KL-1492 by 80.4% and for KL-1507 by 37.58% (Fig. 9, 10: E).
Last but not least, when applied in the highest concentration (150 ȝM) both compounds
decreased electron transport system capacity (ETS-obtained by FCCP successive
titrations). Hence, KL-1492 decreased the uncoupled glutamate/malate-supported
respiration by 49%, and KL-1507 by 39.8% vs. Ctrl, respectively (Fig. 9, 10: D).
As mentioned above, state 2 represents the basal respiration whereas state 4 was
pharmacological induced with oligomycin (Omy) that inhibits the proton entry at the level
of F0-ATPase. Compounds able to stimulate respiratory rates in state 4 (and 2) in a dose-
dependent fashion are known as uncouplers because they disrupt (or uncouple) the link
between substrate oxidation and ADP phosphorylation in ATP. Conversely, mitochondrial
respiration that has been inhibited by inhibitors such as antimycin cannot be released by
the uncouplers [89].
We further tested the effects of KL-1492 and KL-1507 in the presence of sodium 5-
hydroxydecanoic acid (5-HD), a mitoKATP channel inhibitor on mitochondrial respiration.
Our results showed that the effects of both tested compounds were not abolished in the
presence of 5-HD for both CI- and CII-supported respiration (Fig. 11 – 14).

27

Figure 11. HRR studies for CI-supported respiration in the presence of KL-1492 and 5-HD. Results are
expressed in pmol/(s*ml). Values are means ± SEM. *p<0.05 vs. Ctrl, #p<0.05 vs. Ctrl + 5-HD

Figure 12. HRR studies for CII-supported respiration in the presence of KL-1492 and 5-HD. Results
are expressed in pmol/(s*ml). Values are means ± SEM. *p<0.05 vs. Ctrl, #p<0.05 vs. Ctrl + 5-HD

Figure 13. HRR studies for CI-supported respiration in the presence of KL-1507 and 5-HD. Results are
expressed in pmol/(s*ml). Values are means ± SEM. *p<0.05 vs. Ctrl, #p<0.05 vs. Ctrl + 5-HD

28

Figure 14. HRR studies for CII-supported respiration in the presence of KL-1507 and 5-HD. Results
are expressed in pmol/(s*ml). Values are means ± SEM. *p<0.05 vs. Ctrl, #p<0.05 vs. Ctrl + 5-HD
3.2. Mitochondrial H 2O2 production
We also tested the effect of these 2 benzopyrane compounds on the ROS release by
isolated mitochondria respiring in the presence of CI substrates, glutamate-malate, using
the Ampl ex Red a ssay . Our resul ts sh owed a signific ant de crease of mi tochon dri al H 2O2
for KL-1492 when applied at 100 and 150 ȝM (Fig. 15 A) and only in the presence of the
highest concentration in the case of KL-1507, respectively (Fig. 15 B). Accordingly, the
addition of KL-1492 decreased mitochondrial H 2O2 release by 80.3% for 100 ȝM and by
88.1% for 150 ȝM, respectively.
Interestingly, KL-1507, when applied at 100 ȝM, showed a slight increase in ROS
production whereas the highest concentration elicited a significant decrease of H 2O2
release (21.50 ± 2.66 vs. 31.23 ± 1.19 pmol H 2O2/min/mg protein, p < 0.05 vs. Ctrl – Fig.
15A). Our data confirm previous studies reporting on the ability of the mK ATP openers to
protect mitochondria function and structure by suppressing ROS generation during
reoxigenation [90-92].

29
Figure 15. Mitochondrial H 2O2 production in GM respiring mitochondria. Results are expressed in
pmol/mg prot/min. Values are means ± SEM. p<0.05

3.3. In vitro sensitivity to Ca2+-induced mPTP opening
In order to validate the CRC method we firstly performed a positive control with CsA (Fig.
16). As we expected, CsA (1 ȝM) exerted protective effects by decreasing the sensitivity to
Ca2+-induced mPTP opening vs. untreated group (Ctrl).

Figure 16. Calcium retention capacity (CRC) assay: representative traces of
CaCl 2 pulses without (Ctrl) and with 1 ȝM CsA (CsA ) in rat heart
mitochondria. A. CRC data showing a significant increase (p<0,001) of CRC in
the presence of CsA vs. Ctrl. B. CaCl 2 pulses (20 nmol/pulse) were added at 1
min intervals, until mitochondrial Ca2+ release caused by opening of the PTP was
observed.
Our measurements showed an increased sensitivity to Ca2+-induced mPTP opening in the
presence of 150 ȝM of KL-1492 or KL-1507 vs. controls (Fig. 17 and 18).

30

Figure 17. CRC assay: representative traces of CaCl 2 pulses without (Ctrl)
and with 150 ȝM KL-1492. A. In the presence of KL-1492, CRC was
significantly reduced when compared to Ctrl. Values are means ± SEM. *p<0.05.
B. CaCl 2 pulses (20 nmol/pulse) were added at 1 min intervals, until
mitochondrial Ca2+ release caused by opening of the PTP was observed.

Figure 18. CRC assay: representative traces of CaCl 2 pulses without (Ctrl)
and with 150 ȝM KL-1507. A. In the presence of KL-1507, CRC was
significantly reduced when compared to Ctrl. Values are means ± SEM. *p<0.05.
B. CaCl 2 pulses (20 nmol/pulse) were added at 1 min intervals, until
mitochondrial Ca2+ release caused by opening of the PTP was observed.

31 4. Discussion
Our data suggest that these 2 compounds act as inhibitory uncouplers, showing an intrinsic
uncoupling effect with increasing concentrations and inhibitory property when used in the
highest doses. Indeed, starting from 50 ȝM these benzopyran analogues uncoupled the
oxidative phosphorylation of mitochondria respiring on the NAD-dependent substrates
glutamate-malate. Moreover, the uncoupling effect was not abolished by 5-
hydroxydecanoate (5-HD, 50-300 ȝM), the putative selective mK ATP inhibitor, suggesting
that the increase in CI-supported basal respiration was not related to the opening of mK ATP
channels. Our findings agrees with the well-established effects of the mK ATP openers
diazoxide and pinacidil, which act as uncoupling protonophores, mainly when applied in
high concentrations in isolated rodent mitochondrial preparations [25, 30, 58-60, 93] in a
potassium channel-independent manner. Also, our data regarding the effect of 5-HD are in
agreement with the previous reports showing that the mK ATP blocker had no effect on the
activation of the basal mitochondrial respiration by diazoxide and pinacidil applied in
similar concentrations [93, 94].
Furthermore, both compounds significantly decreased state 3 respiration as early
demonstrated for diazoxide [21, 25] and pinacidil [94]. In order to investigate whether the
decrease of state 3 respiratory rate can be assigned to the mK ATP opening, we recapitulated
the experiments in potassium chloride and choline chloride medium (without K+). Since
the effect of the compounds at 150 ȝM on state 3 respiration remained unchanged (data not
shown), no relation with the K+ flux into the matrix can be affirmed. This observation is in
l i n e w i th t h e r e s ul t s r e p o r t e d f o r p i n a c i di l i n t h e s a m e e x p e r i m e n t a l m o d e l by T ol e i k i s '
group [94], who found a K+-independent decrease in state 3 and also in uncoupled
respiration in the presence of complex I respiratory substrates. Of note, the latter effect was
found for our compounds when applied in the maximal concentration.
It has to be mentioned that the most investigated benzopyrane derivate, BMS 191095, was
reported to exert cytoprotective effect against a calcium-ionophore induced injury in a
skeletal muscle cell line C2C12 [95]. This it did by promoting cellular survival, despite an
impaired mitochondrial function, as shown by a decrease in state 3 respiration.
Interestingly, cytoprotection against calcium overload elicited by the specific mK ATP
opener was not reversed in this model by 5-HD, even though the channel inhibitor was
applied in high a concentration (500 ȝM). Whether this might be related to the previously

32 reported inhibition of respiration by high concentrations (100 and 300 ȝM) of 5-HD is not
known yet [96]. Moreover, the beneficial neuroprotective effects of BMS 191095 against
cerebral ischemia could be reversed only when 5-HD was applied in very high millimolar
concentrations [97]. However, it cannot be ruled out that, as shown for diazoxide, the
ability of 5-HD to reverse protection is related to its metabolic effects and does not result
from mK ATP inhibition [98].
Collectively, our data shows a significant uncoupling effect of the two novel benzopyrane
analogues at the concentration of 100 and 150 ȝM, and the inhibition of mitochondrial
respiration when applied in higher concentration (150 ȝM) in isolated rat heart
mitochondria. These effects are most probably not related to the activation of mK ATP
channels but rather suggestive for a protonophoric action – as reported for the prototype
mKATP opener diazoxide in the pioneering studies of Portenhauser in 1971 [99], and more
recently for diazoxide and pinacidil [30, 58].
H owever, s everal issues tha t remain to be cla rified can be regarded as lim ita ti ons of the
p r e s e n t s tu dy . Fi r s tly , th e p r ot on oph or i c a c t i on of t he i nv e s ti g a t e d c o m po un ds h a s t o be
demonstrated (e.g., by investigating the concentration-dependent effect on mitochondrial
swelling). Also, assessing the effect of BSA on the uncoupling effect is worthy since it has
been reported that the uncoupling effect of diazoxide was significantly depressed in the
incubation medium by the lattter [94].
In the past decade the interest of the scientific community largely moved to the modulation
of the mitochondrial permeability transition pore (mPTP) as novel mitochondrial target for
cardioprotection while studies addressing mK ATP channels mainly attempted to elucidate
its structure. However, it has to be mentioned that the selective benzopyran derivate BMS
191095, elicited both antinecrotic [100] and antiarrhythmic [101] protection in the rodent
heart subjected to I/R injury, and still represents an useful tool for basic research [29]. In
this line, a functional crosstalk between mPTP and mK ATP that determined the arrhythmic
vulnerability to oxidative stress has been recently reported [102]. In order to evaluate if
uncoupling can reduce the sensitivity of heart mitochondria to undergo calcium-induced
mPT we tested the effects of both compounds, at the highest concentration (i.e., 150 µM),
on CRC. We showed here that administration of both compounds in RHM isolated from
healthy animals induced an increased sensitivity to Ca2+-induced mPTP opening, results
which are in line with a recent paper reporting a negative effect of respiratory uncoupling
on calcium retention capacity of cardiac mitochondria [56]. As suggested by Morota et al.,

33 a possible explanation for decreased CRC is that "any beneficial effect on heart
mitochondrial CRC by an enlarged matrix volume following increased K+ flux is
counteracted by a respiratory inhibition and a direct negative effect of uncoupling” [56].
Whether the above mentioned effects of the novel mK ATP openers can be recapitulated in
the settings of I/R injury will be further investigated. Indeed, as pointed out in an excellent
review by Brookes in 2005 [61], “uncoupling of mitochondria decreases ROS but
uncoupling of inhibited mitochondria increases ROS” and the characterization of novel
uncoupling agents for tissue protection represents an emerging research field. Also, studies
describing the effects of our compounds on mitochondrial resistance to calcium overload
are required, in line with a recent paper reporting a negative effect of respiratory
uncoupling on calcium retention capacity of cardiac mitochondria [56].

34 PART III. CONCLUSIONS
1. KL-1492 and KL-1507 represent novel benzopyrane analogues able to
modulate mitochondrial respiratory function and the release of
hydrogen peroxide in isolated rat heart mitochondria respiring on complex I
substrates.
2. High-resolution respirometry studies conducted in the presence of the
pharmacological modulators of mitoK ATP revealed both an uncoupling
effect and respiratory inhibition for the highest investigated
concentrations, 100 and 150 ȝM, respectively.
3. When applied in these concentrations, the investigated compounds also
mitigated the H 2O2 release.
4. The highest concentration of both compounds elicited a negative effect on
the calcium retention capacity of isolated mitochondria.
5. sensitivity of mPTP opening was increased by 150 ȝM of both compounds,
suggesting that there are no direct beneficial effects of increased K+ or
H+ flux on heart mitochondrial resistance to mPTP.

35 ORIGINAL FINDINGS AND FUTURE DIRECTIONS
The above presented research contributed to the characterization of two novel compounds
that could be beneficial in cardioprotection at reperfusion via the modulation of
mitochondrial function.
Parts of the results were presented at the Research (TDK) conference for undergraduate
students at the University of Szeged on the 12th of February 2015, as can be seen attached
in the Annex.
Further studies aimed at assessing the effects of these pharmacological agents in the setting
of ischemia/reperfusion injury are warranted.

36 ACKNOWLEDGEMENTS
This work was performed in the Department of Functional Sciences, Chair of
Pathophysiology at the "Victor Babes" University of Medicine and Pharmacy, Romania.
T h i s r e s e a r ch w as s u pp or t e d by a g r a n t of th e Mi ni s t ry of Na t i on al E duc a ti on , C N C S –
UEFISCDI, project number PN-II-ID-PCE-2012-4-0512.
The author wishes to thank Danina Muntean, Oana Duicu and Alexandra Petrus for their
assistance in writing the previous work; Ayko Bresler and Alexander Grimme for helping
with the layout.

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43 ANNEX

44

45

46