Instituto Tecnológico y de Estudios Superiores de Monterrey

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Instituto Tecnológico y de Estudios Superiores de Monterrey

Campus Monterrey

Escuela de Medicina y Ciencias de la Salud

The role of the Mitochondrial Calcium Uniporter in the process of arrhythmogenesis in a murine model of acute catecholamine overload

Disertación presentada por

Felipe de Jesús Salazar Ramírez

sometida a la

Escuela de Medicina y Ciencias de la Salud

como un requisito parcial para obtener el grado académico de

Maestro en

Ciencias Biomédicas

Monterrey Nuevo León, 22 de mayo de 2020

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Dedication

Dedico este trabajo a mis amigos y compañeros de la maestría, de quienes no sólo aprendí a trabajar en equipo, sino también a crecer como persona; a mi esposa, quien me estuvo acompañando durante largas noches de trabajo con paciencia y comprensión; a mi familia, que me mantuvieron con ánimos en todo momento; a los doctores y post-docs del grupo de cardiología, de quienes siempre estuve aprendiendo y con quienes podía contar para resolver mis dudas. Dedicado con cariño al Señor T., Libby y Camila.

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Acknowledgments

This study was performed in the Laboratory of cardiology of the Cardiology and Vascular Medicine Institute in Zambrano-Hellion Medical Center. This research was made possible with the help and guidance of the whole Cardiology team, as their help and advice were sought constantly to solve issues that kept appearing along the way. I would also like to acknowledge the veterinarians in charge of the Tecnológico de Monterrey’s Animal household, who kindly offered their help and capacitation to learn to handle animals properly and treat them with respect, as well as making the long hours of work easier to handle. I want to acknowledge Tecnológico de Monterrey for tuition support and CONACYT for the monthly stipend.

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The role of the Mitochondrial Calcium Uniporter in the process of arrhythmogenesis in a murine model of acute catecholamine overload

By

Felipe de Jesús Salazar Ramírez

Summary

Sudden cardiac death by fulminant ventricular arrhythmias remains a concern in population with cardiac risk. Recently, the mitochondrion has been implied to be a central player in Ca²⁺ mishandling, with its dysfunction leading up to arrhythmogenesis.

A possible starting event that could lead to most changes seen in cardiac disfunction is mitochondrial Ca²⁺ overload. The following research study focuses on demonstrating the effects of mitochondrial Ca²⁺ influx inhibition in arrhythmogenesis. A murine model of acute catecholamine (isoproterenol) overload was treated previously with mitochondrial Ca²⁺ transport inhibitor Ru360. Ru360 treated mice showed a complete abolishment of ventricular tachycardia and ventricular fibrillation. To characterize the possible

mechanisms of action, heart mitochondria were isolated and mitochondrial function was assessed. Mitochondrial Ca²⁺ transport inhibition preserved mitochondrial function and membrane integrity as demonstrated by a higher respiratory control and calcium retention capacity when compared to isoproterenol-treated mice which appears to be caused by a reduced oxidative stress as a trend to preserve reduced thiol groups was shown. Given the positive results obtained in abolishing ventricular arrhythmias by inhibiting

mitochondrial Ca²⁺ transport, it is precise to continue the characterization of the

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mechanisms by which this therapy exerts its effects. To fully demonstrate its efficacy and characterize its mechanism of action may lead up to a new therapeutic target and therapy that could set the bases to clinical research in the near future.

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Chapter 1- Problem statement

Arrhythmias can be defined as any disturbance in the normal electrical sequence of the heart. These disturbances may cause the electrical impulse to travel slowly, rapidly or in an erratic manner. Few studies calculate the overall burden of arrhythmias.

Incidence has been reported to be about 2.35% in the general population of the UK (Khurshid et al., 2018) while in Mexico only data for auricular fibrillation exists, which is estimated to be about 2% of the general population (Lara-Vaca, Cordero-Cabra,

Martínez-Flores, & Iturralde-Torres, 2014). This data coincides with what’s reported globally, which is estimated to be around 1-4% (Zulkifly, Lip, & Lane, 2018). Even though it may seem as a small percentage, electrical abnormalities appear in up to 39% of patients with cardiopathies (Vazquez Ruiz de Castroviejo et al., 2005). Within this

population, sudden cardiac death constitutes an important cause of mortality. Sudden cardiac death is defined as when the death of a patient occurs, most commonly by a fatal ventricular arrhythmia, within one hour of the onset of symptoms when there is a witness and within 24 hours of last being seen alive when no witness is available (Adabag, Luepker, Roger, & Gersh, 2010). This is especially concerning in high risk populations such as heart failure patients, in which the incidence of sudden cardiac death reaches approximately 15% per year and whose population keeps growing continuously as it is the final outcome of almost all cardiovascular pathologies (Lee et al., 2011; Srinivasan &

Schilling, 2018). Again, there are no statistics available reporting incidence of sudden cardiac death in Mexico, although it has been documented to be responsible of 20-30% of all cardiac deaths worldwide and about 7-18% of total mortality in the USA (Rodríguez- Reyes et al., 2015). Analyzing the process of arrhythmogenesis at a cellular level is thus

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of vital importance to better understand the underlying mechanisms that lead up to its development and elucidate key new potential therapeutic targets to prevent it.

In cardiac tissue very special attention is focused on calcium handling since it participates in many cellular processes such as contraction, energy production and cellular signaling (Bers, 2002; Fernandez-Sada et al., 2014; Kwong et al., 2015). Knowing all the processes in which calcium is involved, it’s no surprise that its mishandling is part of a wide range of pathologies such as arrhythmogenesis (Aistrup, Balke, & Wasserstrom, 2011). Recently, special attention has been brought to the interconnection that lies between the sarcoplasmic reticulum (SR) and mitochondria (Santulli, Xie, Reiken, & Marks, 2015). The SR, as the main cellular Ca²⁺ reservoir, and the mitochondria´s, as the prime energy provider, close relationship provide an adequate cellular Ca²⁺ handling, which translates into contractile force and an efficient cytosolic Ca²⁺ removal system. It is no surprise components of the SR and mitochondria involved in Ca²⁺ handling have been described to be dysfunctional in pathologies with a high risk of developing arrhythmias (Federico, Valverde, Mattiazzi, & Palomeque, 2019; van Opbergen, den Braven, Delmar, & van Veen, 2019). Notably, the ryanodine receptor (RyR) and SERCA protein complexes seem to be related to the mitochondrial Ca²⁺

uniporter (MCU)-driven mitochondrial Ca²⁺ overload (Santulli et al., 2015). In this sense, several mechanisms, such as oxidative stress might increase Ca²⁺ concentration in the SR-mitochondria microdomain, which in turn would produce an energetic debacle, that further increases cytosolic Ca²⁺ overload and the generation of abnormal depolarization with subsequent arrhythmogenesis (Santulli et al., 2015).

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In the present study, a mitochondria-targeted strategy to prevent arrhythmias was assessed. As it has been shown that preventing Ca²⁺ overload may be effective in

preventing acute catecholamine-induced arrhythmogenesis. However, more studies are needed to prove the effects that preserving mitochondrial function has in the context of cardiac pathologies with a high risk of developing arrhythmias.

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Chapter 2- Theoretical framework

Note: The following chapter is part of a review paper prepared for the research topic:

Mitochondrial Remodeling and Dynamic Inter-Organellar Contacts in Cardiovascular Physiopathology. Hosting Journal/Specialty: Frontiers in Cell and Developmental Biology - Cellular Biochemistry.

Mitochondrial and Sarcoplasmic Reticulum interconnection as a trigger of arrhythmogenesis

Felipe de Jesús Salazar-Ramírez¹ and Gerardo García-Rivas^1,2,*

1Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Monterrey, México.

2Centro de Investigación Biomédica, Hospital Zambrano-Hellion, San Pedro Garza García, México.

*Correspondence: [email protected]

Keywords: mitochondria, calcium, arrhythmia, sarcoplasmic reticulum, interconnection

Abstract

Arrhythmogenesis is a complex process as it involves many subcellular components that can be analyzed as the central player of inducing abnormal heart contraction. Recently, special attention has been brought to the interconnection that lays between the

sarcoplasmic reticulum and mitochondria. SR, as the professional cellular Ca²⁺ reservoir, and mitochondria as the prime energy provider, are intimately related to provide an adequate Ca²⁺ release, which translates into contractile force, and an efficient cytosolic Ca²⁺ removal system, which translates into relaxation. It is no surprise that components of the SR and mitochondria involved in Ca²⁺ handling have been found to be

dysfunctional in patients with a high risk of developing arrhythmias. Particularly, RyR2 and SERCA seem to be related with mCU-driven mitochondrial Ca²⁺ overload and could be relevant players involved in Ca²⁺ handling and malfunctions that triggers arrhythmias and sudden death. Several possible mechanisms that increase in SR-mitochondrial Ca²⁺

flux, such as Ca²⁺/calmodulin-dependent protein kinase II, reactive oxygen species and oxidation of the RyR2, SERCA and mCU can increase Ca²⁺ concentration in the SR- mitochondria microdomain which in turn would produce mitochondrial Ca²⁺ overload and mitochondrial dysfunction. This would lead to an energetic debacle that further increases cytosolic Ca²⁺ overload and generation of DADs or EADs in cardiomyopathy with possible subsequent arrhythmogenesis. In this review a new point of view is

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proposed. A new approach where the sarcoplasmic reticulum is not the sole central player, but instead is part of a duo with the mitochondria in maintaining Ca²⁺ homeostasis by an intimate communication between the organelles. It is then this failed

communication that helps explain the development of Ca²⁺ mishandling with subsequent risk of developing arrhythmias in cardiac patients.

Abbreviations

SR- Sarcoplasmic reticulum Ca2+- Calcium

mCU- Mitochondrial calcium uniporter SERCA- SR Ca2+-ATPase

RyR- Ryanodine Receptor

DAD- Delayed afterdepolarization EAD- Early afterdepolarization AP- Action potential

NCX- Na+/Ca2+ exchanger

ECC- Excitation-contraction coupling ECEC- Excitation-contraction-energetics coupling

ΔΨm- Mitochondrial membrane potential ROS- Reactive oxygen species

IMM- Inner mitochondrial membrane mPTP- Mitochondrial permeability transition pore

FUNDC1- FUN14 domain containing 1 VAPB- Vesicle-associated membrane protein-associated protein B/C

PTPIP51- protein tyrosine phosphatase- interacting protein 51

ERMES complex- ER-mitochondria tethering protein complex

PDZD8- PDZ domain-containing protein 8

INF2- Inverted formin 2

mNCX- Mitochondrial Na+/Ca2+

exchanger

GSH- Reduced glutathione AF- Atrial fibrillation

mROS- Mitochondrial reactive oxygen species

AMPK- AMP-activated protein kinase UCP3- Uncoupling protein 3

UCP2- Uncoupling protein 2 ETC- Electron transport chain

CAMKII- Ca 2+/calmodulin-dependent protein kinase II

ECG- Electrocardiogram AAV- adeno-associated vector EF- Ejection fraction

LV- Left ventricle PLB- Phospholamban CsA- Cyclosporine A

CPVT- Catecholaminergic polymorphic ventricular tachycardia

VT- Ventricular tachycardia VF- Ventricular fibrillation

DMD- Duchenne muscular dystrophy

Introduction

Arrhythmias can be defined as any disturbance in the normal electrical sequence of the heart. These disturbances may cause the electrical impulse to travel slowly, rapidly or in an erratic manner. Few studies calculate the overall burden of arrhythmias. Incidence has been reported to be about 2.35% in the general population of the UK (Khurshid et al., 2018) while in Mexico only data for auricular fibrillation exists, which is estimated to be about 2% of the general population (Lara-Vaca et al., 2014). This data coincides with what’s reported globally, which is estimated to be around 1-4% (Zulkifly et al., 2018).

Even though it may seem as a small percentage, electrical abnormalities appear in up to 39% of patients with cardiopathies (Vazquez Ruiz de Castroviejo et al., 2005). Within this population, sudden cardiac death constitutes an important cause of mortality. Sudden

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cardiac death is defined as when the death of a patient occurs, most commonly by a fatal ventricular arrhythmia, within one hour of the onset of symptoms when there is a witness and within 24 hours of last being seen alive when no witness is available (Adabag et al., 2010). This is especially concerning in high risk populations such as heart failure patients, in which the incidence of sudden cardiac death reaches approximately 15% per year and whose population keeps growing continuously as it is the final outcome of almost all cardiovascular pathologies (Lee et al., 2011; Srinivasan & Schilling, 2018).

Again, there are no statistics available reporting incidence of sudden cardiac death in Mexico, although it has been documented to be responsible of 20-30% of all cardiac deaths worldwide and about 7-18% of total mortality in the USA (Rodríguez-Reyes et al., 2015). Analyzing the process of arrhythmogenesis at a cellular level is thus of vital importance to better understand the underlying mechanisms that lead up to its development and elucidate key new potential therapeutic targets to prevent it.

In cardiac tissue very special attention is focused on Ca²⁺ handling since it participates in many cellular processes such as contraction, energy production and cellular signaling (Bers, 2002; Glancy & Balaban, 2012). Knowing all the processes in which Ca²⁺is involved, it’s no surprise that its mishandling is part of a wide range of pathologies such as cardiac hypertrophy (Power, Hickey, Crossman, Loiselle, & Ward, 2018; Tham, Bernardo, Ooi, Weeks, & McMullen, 2015), heart failure (Bers & Despa, 2006), dilated cardiomyopathy (Law, Cohen, Martin, Angulski, & Metzger, 2020) and

arrhythmogenesis (Aistrup et al., 2011; Eisner, Caldwell, Kistamas, & Trafford, 2017).

The sarcoplasmic reticulum (SR) is the organelle in charge of storing and releasing Ca²⁺

to the cytosol. The opening of the ryanodine receptor (RyR) occur after an initial small amount of Ca²⁺ passes through L-type Ca²⁺ channels in the sarcolemma as a response to membrane depolarization during the action potential (AP) (Bers, 2002). The sudden increase in cytosolic Ca²⁺ levels activates the myofibrils in sarcomeres and contraction can occur. After Ca²⁺ has been released and contraction has taken place, Ca²⁺ then needs to be removed from the cytosol in order to reestablish the cellular baseline environment for another AP to be able to take place. This is done in its majority by the SR Ca²⁺- ATPase (SERCA), which pumps about 70% of total cytosolic Ca²⁺ back into the SR (Bassani, Bassani, & Bers, 1994). The sarcolemmal Na⁺/Ca²⁺ exchanger (NCX) also removes a small amount by introducing 3 Na⁺ ions per Ca²⁺ extruded into the

extracellular space (Bers, 2002). This process that starts with membrane depolarization that causes a sudden increase in cytosolic Ca²⁺ levels that activates the contractile apparatus is known as excitation-contraction coupling (ECC). Under pathologic conditions, depolarization of the sarcolemma may not elicit the needed Ca²⁺ release or not elicit one at all, with the subsequent consequence of a less effective contraction. More than that, a RyR may release Ca²⁺ without an appropriate signal, possibly propagating more Ca²⁺ release by other RyR through the rest of the cardiomyocyte and evoking an unorganized contraction (Kistamás et al., 2020). Much attention has been put in

describing the structural changes that the SR and its components suffers (Kubalova et al., 2005), often correlating with severity of the disease but also opening more questions as to why those changes takes place in the first place. This approach has proven useful in better

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understanding the molecular mechanisms of the disease. However, the cause of these changes is not yet fully described or understood. Unraveling the causes of these changes are essential for designing new therapies to target the root of the problem as a therapy that merely targets its effects may only provide a temporal solution. It might be that another important player, the mitochondria, needs to be considered along with the SR to better understand Ca²⁺ mishandling. In cardiac cells, mitochondria and the SR are interconnected and form a structure along the cardiomyocytes. The close opposition between mitochondria and the SR creates Ca²⁺ microdomains that contribute to ECC, where the mitochondrial Ca²⁺ transport plays a significant role. The mitochondrial-SR

"bridge" is established through several proteins from both organelles (De la Fuente &

Sheu, 2019). During the rise of cytosolic Ca²⁺ concentration by the opening of the RyRs, a small amount is taken up by the mitochondria into its matrix through a Ca²⁺ channel in the internal mitochondrial membrane, the mitochondrial Ca²⁺ Uniplex (mCU) (previously called mitochondrial Ca²⁺ uniporter). RyR and mCU are co-localized in the

mitochondria-SR contact sites (Kohlhaas & Maack, 2013). Coupled Ca²⁺ transport

between mitochondrial-SR regulates several processes involved in arrhythmogenesis such as Ca²⁺ handling, mitochondrial energetics, redox homeostasis, among others. In this review we will focus on describing the cross signals that happens between these two organelles and the signaling involved in a pathologic setting that might trigger arrhythmias.

The Mitochondria-SR interconnection

About 90% of the heart’s ATP is produced through oxidative phosphorylation in mitochondria (Harris & Das, 1991). Given that the heart is beating continuously, that muscle contraction is an extremely ATP-demanding process and that mitochondria constitute about 25% of the volume of human cardiomyocytes, even more so in rats and mice (~30%) (Schaper, Meiser, & Stammler, 1985), it reflects the organelle’s relevance in cardiac tissue. Rising of cytosolic Ca²⁺ levels during the AP stimulates Ca²⁺ transport into the mitochondrial matrix. This rise of intramitochondrial Ca²⁺ concentration

activates dehydrogenases involved in Kreb’s cycle which in turn supplies the electron transport chain with NADH⁺ and FADH2 to meet the cell’s energetic demand

(Fernandez-Sada et al., 2014; Glancy & Balaban, 2012; McCormack & Denton, 1990). In this sense, in cardiac cells Ca²⁺ signal is coupled with the energy demand through a process called the excitation-contraction-energetics coupling (ECEC). This process links Ca²⁺ handling and the contraction of the cardiomyocyte with ATP production in

mitochondria (Maack & O'Rourke, 2007). Mitochondrial Ca²⁺ transport occurs when SR release Ca²⁺ in the vicinity of mCU. This channel is a complex made up by a pentamer forming the pore section and several regulatory subunits (Fan et al., 2020) which opens the channel only at high Ca²⁺ concentrations in the cytosol, using the mitochondrial membrane potential (ΔΨm) as the driving force for transportation (Bertero & Maack, 2018). The ATP synthesized is then used by SERCA to remove Ca²⁺ from the cytosol and

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binds to the contractile apparatus, replacing ADP at the myofibrils, to enable relaxation.

Yet, ATP production is far from being the organelle’s sole function. Additionally, since mitochondria is localized near the SR and T-tubules, it’s possible for the mitochondria to not only to receive Ca²⁺ signaling, but also indirectly send signals back, modifying crucial proteins involved in the AP development through means such as reactive oxygen species (ROS) or by starting a signaling cascade that may have a wide range of effects.

For instance, high mitochondria Ca²⁺ levels can lead to mitochondrial Ca²⁺ overload- related features readily seen in cardiac cells such as ΔΨm depolarization and reduced ATP synthesis. Excess mitochondrial Ca²⁺ levels bind to sites on the internal

mitochondrial membrane (IMM), promoting the formation of the mitochondrial permeability transition pore (mPTP), and possibly perpetuating its presence in cardiac pathologies. The mPTP opening results in loss of cristae surface and swelling of the IMM, and finally followed by irreversible mitochondrial dysfunction that trigger cell death (Hurst, Hoek, & Sheu, 2017). This crosstalk between the SR and mitochondria is vital for an adequate heart contraction to take place. Disturbances in this signaling may cause deleterious effects that can end up in ECEC-triggered cell death.

The architecture of a cardiomyocyte has been previously described (Eisner et al., 2017) and emphasis is made about in the proximity of T-tubules, where L-type Ca²⁺channels reside, with the terminal cisternae of the SR, where RyR are more concentrated. The approximate distance between both structures have been calculated to be only about 15 nm (Scriven, Asghari, & Moore, 2013). This is what enables the two structures to react to each other’s activation. Either as the RyR opening from an initial activation of L-type Ca²⁺ channels in T-tubules or by L-type Ca²⁺ channels closing when a high concentration of Ca²⁺ is released from the RyR. This unit comprised of a T-tubule with its

corresponding terminal cisternae of the SR is named dyad and is considered as the functional unit of the heart in charge of coupling excitation and contraction. Nonetheless, the mitochondria could also be considered as part of this functional unit as it also plays a role in connecting stimulation with a proper contractile response. To make this possible, the SR and the mitochondria have a constant communication to adequately respond to energetic demands in various situations.

Proximity enables crosstalk

Since long ago, SR-mitochondria interconnections have been described. Fluorescence and electron microscopy have been used to show physical interaction between organelles, with protein-like structures linking both membranes (De la Fuente & Sheu, 2019). Protein structures between the mitochondria and the SR have been already described

elsewhere(Giorgi, Marchi, & Pinton, 2018; Lopez-Crisosto et al., 2017; Martinvalet, 2018). Several structures have been found to create bridges between the organelles, securing proximity (Figure 1). The complexity of SR-mitochondria bridging proteins is high, such that it may be case that removal of individual structures may be compensated by other components. Describing the interaction of each component found to link both organelles in detail is out of the scope of this review, so a small description will be made.

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The first of such structures is the Ca²⁺ channel IP3R1, which is joint to the mitochondrial ion channel VDAC through protein GRP75. This communication enables fast movement of Ca²⁺ions from the SR into the mitochondrial intermembrane space when IP3 is released through the PKC pathway (Lopez-Crisosto et al., 2017). Similarly, IP3R2 has been described to bind with FUN14 domain containing 1 (FUNDC1) to modulate SR Ca²⁺ release (Wu et al., 2017). VDAC has also been described as having physical interactions with the RyR, which coupled with mCU co-localization with the RyR helps explain how mitochondrial Ca²⁺ transport is possible (Kohlhaas & Maack, 2013). Other structures involved in maintaining connection are the SR vesicle-associated membrane protein-associated protein B/C (VAPB), whose function is not fully understood although it has been shown regulate Ca²⁺ transport between both organelles (De Vos et al., 2012), and the mitochondrial protein tyrosine phosphatase- interacting protein 51 (PTPIP51), seem to have more structural functions (Stoica et al., 2014). The ER-mitochondria tethering protein complex (ERMES complex), is a protein structure characterized in yeast that bridges both organelles as well as having diverse biological functions. Although no homologue structure has been described in mammals, recently an orthologue of one of its components, PDZ domain-containing protein 8 (PDZD8) has been described (Giorgi et al., 2018). Other structures, such as Mitofusin2, a protein involved in mitochondrial dynamics, have also been described as being able to form dimers that bridge both

organelles (de Brito & Scorrano, 2008), presumably to organize mitochondrial dynamics.

Similarly, SR protein inverted formin 2 (INF2) serve as anchor for actin filaments to reach the mitochondria and this way make scaffolding for mitochondrial constriction in mitochondrial dynamics (Korobova, Ramabhadran, & Higgs, 2013; Manor et al., 2015).

These connections help maintain the close gap between mitochondria and the SR.

Intermyofibrillar mitochondria have been measured as close as 33 nm from the RyR in the SR and as far as 188nm (Ramesh, Sharma, Sheu, & Franzini-Armstrong, 1998). This enables the existence of a microdomain where tight communication can take place. For instance, cellular Ca²⁺ concentration varies from about 100nM-500nM globally between resting and peak concentrations in the AP (Bers, 2002) but within the dyadic cleft, Ca²⁺

concentration can get as high as 100 µM at the periphery when the RyR2 releases Ca²⁺

from the SR stimulated by L-type Ca²⁺ channels (Langer & Peskoff, 1996). These high levels of Ca²⁺ are maintained for about 10ms, affecting a region of about 2 µm, although not in the same concentrations, before descending as Ca²⁺ diffuses to other cellular regions (Cheng & Lederer, 2008). This places the mitochondria well within reach to access high local Ca²⁺ concentration. Even though 10ms may not seem as much, this is enough time for Ca²⁺ to be transported through the IMM by the mCU into the

mitochondrial matrix. Two models describing the mitochondrial response to changes in cytosolic Ca²⁺ levels have been described (De la Fuente & Sheu, 2019). The first one states that mitochondrial Ca²⁺ concentration increases slowly and gradually with a faster AP firing rate until influx and efflux are balanced completely and a new steady state is achieved. However, slow changes in mitochondrial Ca²⁺ concentration may not be able to stimulate ATP production fast enough to supply immediate metabolic needs. The other model describes the mitochondria as having the ability to fully sense rapid cytosolic Ca²⁺

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changes, presenting with oscillations in a beat to beat basis. This would imply that mitochondria have the capacity to rapidly internalize but also extrude Ca²⁺ ions.

Somehow consolidating both models, a mitochondrial Ca²⁺ transient was described by Lu et al.(Lu et al., 2013) which have some key differences when compared to its cytosolic counterpart. Mainly, as firing rate of the AP becomes faster there is a decrease in

amplitude that maintains a more stable concentration throughout the whole AP by slowly rising diastolic Ca²⁺ concentration even though a faster decline was also noted compared to baseline after stimulating with the catecholamine analogue, isoproterenol (ISO). It is estimated that this happens because mitochondria have slower structures to pump Ca²⁺

out into the cytosol, mainly the mitochondrial Na⁺/Ca²⁺ exchanger (mNCX), as opposed to the combined pumping force of SERCA in the SR and the sarcolemmal NCX. While it has been calculated that the percentage of cellular Ca²⁺ taken up by the mitochondria is only around 1-2% of total cytosolic Ca²⁺ (Bassani et al., 1994) this can have major effects in excitation contraction coupling. The main reason being that, by increasing

mitochondrial Ca²⁺ concentration, dehydrogenases from the Krebs cycle change to a more active form which in turn produce more high energy products (NADH⁺ and FADH2) for the electron chain transport to use as substrates to generate mitochondrial membrane potential (ΔΨm) and subsequent ATP synthesis under a more energy

demanding state, such as when adrenergic stimulation takes place (Fernandez-Sada et al., 2014; Kwong et al., 2015). This ATP is then transported to the cytosol where it is used by the sarcomere to relax its myofibrils and, equally as important, is also used by a wide range of pumps to maintain ion balance. One of those pumps is SERCA, which returns most Ca²⁺ ions, about 70% (Bassani et al., 1994), into the SR after the AP finishes and reestablishes basal Ca²⁺ concentration. This is just one example by which the

mitochondria can communicate back to the SR under demanding and stressful conditions.

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Mitochondrial Ca²⁺ mishandling as a trigger of cardiac arrhythmia

High cytosolic Ca²⁺concentration is what triggers mitochondrial Ca²⁺ transport by the mCU. Under homeostatic conditions this process is finite, and Ca²⁺ can slowly be transported back into the cytosol by the mNCX. However, if Ca²⁺ can’t be extruded out of the mitochondria in time before more Ca²⁺ enters by the mCU then Ca²⁺ overload ensues. This high mitochondrial Ca²⁺ concentration then causes higher ROS production, loss of mitochondrial membrane integrity with subsequent loss of ΔΨm and opening of the mitochondrial permeability transition pore (mPTP). This last event is the first step in the signaling cascade for programmed cell death which can cause further alterations in hearts rhythmicity and organization at a tissual level. It may be a subject of debate to whether SR or mitochondrial dysfunction is what causes the initial Ca²⁺ overload.

Nonetheless, it is a safe bet to argue that what causes this initial malfunction is constant adrenergic stimulation.

Catecholamines are tyrosine-derived hormones synthesized in the adrenal medulla and released under stress signals. These hormones have a wide variety of effects in the whole body that, in general terms, increases catabolism and reduces anabolism by binding to its adrenoreceptor. In myocardial tissue, this occurs by a g-protein coupled receptor that activates adenylate cyclase which turns ATP into cAMP that then activates PKA. PKA

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then phosphorylates proteins involved in cardiac contraction, increasing their function.

Such proteins are: L-type Ca²⁺ channels, which stimulates opening with subsequent Ca²⁺

entry to the cell; the RyR receptor, which sensitizes the channel to release Ca²⁺ under stimulation; SERCA-inhibiting protein PLB, which in its phosphorylated state separates from SERCA thus activating its pump function to remove Ca²⁺ faster and more

efficiently from the cytosol; and troponin I, which causes faster relaxation of the sarcomere by a faster dissociation from Ca²⁺ (R. Zhang, Zhao, Mandveno, & Potter, 1995). All these effects increase the myocardium’s batmotropism and inotropism as well as the pacemaker’s chronotropism. At a cellular level, this translates into a faster release and removal of Ca²⁺ from the cytosol as well as a higher Ca²⁺ content in the SR. Having faster and more frequent APs means cytosolic Ca²⁺ concentration remains high for longer periods of time, which can then be transported into the mitochondria by the mCU.

The cytosol may have the appropriate adaptations to deal with higher Ca²⁺ levels like SERCA and the NCX stated before, but the mitochondria only has the mNCX to deal with this higher Ca²⁺ concentration. As it has been described above, a higher frequency of APs reduces mitochondrial Ca²⁺ transient’s amplitude and stabilizes mitochondrial Ca²⁺

concentration by slowly increasing diastolic Ca²⁺ content, reflecting the organelle’s low capacity to pump Ca²⁺ back into the cytosol. Normally, the mitochondria have means by which to withstand the increased ROS production associated with an increased Ca²⁺

content. These protective mechanisms are composed largely by its reduced glutathione reserve (GSH), mitochondrial superoxide dismutase, mitochondrial catalase and mitochondrial MTHFD, an enzyme that degrades glycine while synthetizing NADPH, which reconstitutes oxidized glutathione (Munro & Treberg, 2017). However, under excessive adrenergic stimulation, as seen in patients with HF where basal catecholamine levels are increased compared to healthy people (Viquerat et al., 1985), the protective mechanisms can be overrun and the once beneficial process to activate enzymes to keep up with the cell’s metabolic demand turns into the trigger for increased ROS production, loss of ΔΨm, mitochondrial dysfunction, mPTP opening and cell death. Not only that, the effects of mitochondrial Ca²⁺ overload reach out to other organelles and structures as well further perpetuating Ca²⁺ dysregulation. Such effects have already been described in the progression of HF in a model of post-ischemic myocardial infarction. Diastolic Ca²⁺ leak from the RyR was associated with mitochondrial dysfunction and further Ca²⁺

mishandling, which could be prevented either by genetic mutation of the RyR that protected against diastolic Ca²⁺ leak or by enhancing mitochondrial antioxidant activity.

(Santulli et al., 2015). Similarly, in an atrial fibrillation (AF) model by tachypacing atrial cardiomyocytes, mitochondrial dysfunction was found, characterized by increased stress chaperones and lower ΔΨm, ATP production and respiration. These findings were

corroborated in the clinical setting in AF patient biopsies, which showed similar changes.

Dysfunction attenuation was possible when Ca²⁺ overload was prevented by either inhibiting or downregulating mCU (Wiersma et al., 2019). Correlation between exacerbated RyR Ca²⁺ and mitochondrial dysfunction was yet again found in

cardiomyocytes of a CPVT murine model under adrenergic stimulation. Increased RyR Ca²⁺ release was associated with increased mitochondrial ROS (mROS) as well as a

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depolarized matrix. Furthermore, this mROS release was associated with RyR oxidation and further Ca²⁺ release. ROS scavenging or decreased mitochondrial Ca²⁺ transport was shown to attenuate the arrhythmogenic phenotype (Hamilton et al., 2020).

Mitochondrial energetic debacle as a trigger of cardiac arrhythmia

First, Ca²⁺ overload increases ROS production which in turn induces protein malfunction, through direct and indirect protein post-translational modification, within the

mitochondria’s Krebs cycle and electron transport chain that ultimately translates into a reduced ΔΨm and ATP production. This energetic depletion can then affect structures that depend on ATP for proper functioning. An obvious example of such protein is SERCA, which needs ATP to transport Ca²⁺ into the SR during myocardium relaxation.

Impairing SERCA’s function reduces SR Ca²⁺ content while increasing the cytosol’s diastolic Ca²⁺ concentration. Another example of an ATP driven protein function is sarcKATP, a potassium ion channel on the sarcoplasmic membrane that opens upon a low ratio of ATP/ADP. This channel is normally inhibited by the high ATP/ADP ratio and, once opened, leads to an efflux of potassium which in turn hyperpolarizes the cell and reduces its excitability and AP duration. It is thought that normally this acts as a safe switch to reduce contractility while the cardiomyocyte replenishes its ATP pool to prevent further damage (Nakaya, 2014). However, in the context of basal mitochondrial disfunction caused by Ca²⁺ overload by a constant adrenergic stimulation, this reduction in batmotropism predisposes the cell to arrhythmogenesis as it may not depolarize properly to an AP of a neighboring cell which would throw off the organ’s organized electric transmission. Recently, AMP-activated protein kinase (AMPK) activated by low ATP levels was found to phosphorylate mCU to allow for a greater flow of Ca²⁺ to enter the mitochondria in mitotic progression (H. Zhao et al., 2019). Even though this has only been recently described in cell undergoing mitosis, it is possible that a similar scenario happens in cardiac tissue. If low ATP concentration can increase Ca²⁺ flow into the mitochondrial matrix, then under chronic low ATP concentrations Ca²⁺ overload can ensue more easily. Moreover, uncoupled mitochondria have been demonstrated to cause AP duration and Ca²⁺ transient duration and amplitude alternans, which are related to generation of ventricular fibrillation. Rabbit hearts submitted to global ischemia or FCCP administration, a mitochondrial uncoupler, were found to have similar effects in inducing AP duration and Ca²⁺ transient alternans (Smith, Visweswaran, Talkachova, Wothe, &

Tolkacheva, 2013). Thus, low ATP synthesis is a possible mechanism for ventricular arrhythmia generation in conditions of mitochondrial stress.

Similarly, impaired cardiac metabolism has been associated with several other cellular changes that predispose to arrhythmia generation. ATP, while mainly used by pumps and contractile machinery, is also used as a second messenger. ATP by itself can bind with RyR2 or Mg²⁺, a RyR inhibitor, and lower the Ca²⁺ threshold needed to elicit a Ca²⁺ - induced- Ca²⁺ release (Fill & Copello, 2002; Meissner, 2004). Low ATP would mean RyR inhibition with a subsequent deficient Ca²⁺ release. An impaired metabolism also increases phosphate levels, which coupled with low ATP can inhibit the sarcolemmal

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Na⁺/K⁺ATPase. This in turn increases cytosolic Na⁺ concentration, which can then stimulate Ca²⁺ extrusion from the mitochondrial matrix through the mNCX, further inhibiting ATP synthesis, and increasing cytosolic Ca²⁺ levels by inhibiting the sarcolemmal NCX (Yang, Kyle, Makielski, & Dudley, 2015)

Interestingly, mitochondria uncoupling also serves as a protection mechanism against cardiac injury and potential arrhythmias through preservation of mitochondria ATP synthesis and decreased ROS generation. Uncoupling proteins have demonstrated to confer protection against cardiac ischemia/reperfusion injury and ischemia-induced arrhythmias in an in vivo model of left coronary artery occlusion (Ozcan, Palmeri,

Horvath, Russell, & Russell, 2013). Uncoupling protein 3 (UCP3) knockdown mice were found to have decreased ATP content, increased AMP/ATP ratio and increased ROS production compared to wild type mice. This was associated with a higher incidence of ischemia-induced arrhythmias, which was not prevented with ischemia preconditioning.

Another uncoupling protein, UCP2, have also been shown to provide protection against Ca²⁺ induced arrhythmias. It seems that UCP2 modulates mitochondrial Ca²⁺ transport and L-type Ca²⁺ current, which has the potential to reduce AP duration significantly in UCP2 KO mice. These electrophysiological changes make UCP2 KO mice more susceptible to arrhythmias upon a stress test (Larbig et al., 2017).

Mitochondrial redox imbalance as a trigger of cardiac arrhythmia

Mitochondria are also known as producers of ROS. ROS are produced when electrons flowing through the electron transport chain (ETC) react in a premature fashion with oxygen before reaching complex IV where they are transported to oxygen and hydrogen to form. Reacting earlier makes radical superoxide, which can then be turned into hydrogen peroxide by mitochondrial superoxide dismutase, or into hydroxyl radical if it encounters a metal with which to react (Alfadda & Sallam, 2012). These byproducts of the electron transport chain, while having a relatively short half-life, can diffuse to nearby cellular compartments, having various effects on a wide range of proteins. ROS

production is directly associated with the activity of the ETC and is inversely proportionate to mitochondrial membrane integrity (Shadel & Horvath, 2015). This means that when mitochondria are activated or damaged, more ROS are produced. Given again, the microdomain that the mitochondria have with the SR, proteins such as the RyR and SERCA are two possible sites of protein oxidation as well as other proteins

implicated in the AP propagation that also reside within or near the dyadic cleft, all of which can affect cellular Ca²⁺ handling.

ROS produced as a result of Ca²⁺ overload doesn’t solely have local effects (Hamilton et al., 2018). These ROS can travel from the mitochondria to nearby organelles and

structures. It has been reported that mitochondrial ROS oxidize the RyR in the context of mitochondrial Ca²⁺ overload (Hamilton et al., 2018; Hamilton et al., 2020). Normally, the RyR senses luminal Ca²⁺ using the help of SR structural proteins junctin, triadin and calsequestrin. Junctin and triadin act as connecting proteins to calsequestrin, which has the property to bind Ca²⁺. Calsequestrin is thought to bind to junctin and triadin,

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inhibiting RyR opening(Györke & Terentyev, 2008). When Ca²⁺ is pumped by SERCA into the SR, calsequestrin then binds with Ca²⁺. This lowers free Ca²⁺ concentration and enables the SR to store more Ca²⁺ than it would be able to do otherwise. Equally as important, Ca²⁺ binding to calsequestin causes polymerization between calsequestin monomers, which are no longer able to bind with junctin and triadin, thus no longer inhibiting RyR. This acts as a luminal Ca²⁺ sensor that permits RyR opening only when SR Ca²⁺ stores have been replenished. Similarly, increased SR Ca²⁺ content have been shown to promote stochastic RyR opening, causing Ca²⁺ sparks (Györke & Terentyev, 2008). Again, its is thought that this happens because an even greater amount of

calsequestrin polymerize and detach from junctin and triadin, further sentizing the RyR by its luminal side. It is hypothesized that this normally regulates the amount of SR Ca²⁺

content to stay within certain parameters. In the case of oxidized RyR, however,

stochastic opening occurs by reducing the SR luminal Ca²⁺ threshold needed to activate the RyR. Not only that, ROS have been described as also being able to oxidize SERCA and impair its functionality (Balderas-Villalobos et al., 2013). L-type Ca²⁺ channels are another possible ROS target. These channels can suffer from glutathionylation, which in increases Ca²⁺ influx upon activation and, even though it is a reversible type of oxidation, constant mROS may maintain this protein state (Johnstone & Hool, 2014). CAMKII is yet another possible target for mROS. This protein is normally activated with Ca²⁺

binding and is in charge of phosphorylating various proteins, one of them which is the RyR. Oxidized CAMKII is known to phosphorylate without needing Ca²⁺ to activate it.

Thus, oxidized CAMKII phosphorylates the RyR lowering even more the luminal Ca²⁺

threshold needed for stochastic opening (Ai, Curran, Shannon, Bers, & Pogwizd, 2005).

Lastly, there have been recent research in which under experimental conditions it is described that the mCU itself can suffer from glutathionylation. Glutathionylation increases the channel’s affinity to Ca²⁺, which now transports Ca²⁺ even at low cytosolic concentrations(Dong et al., 2017). A more sensible mCU transporting more Ca²⁺ into the mitochondrial matrix lead up to a positive feedback of mitochondrial Ca²⁺ overload producing more ROS-induced ROS release within the mitochonria.

All protein malfunction leads to Ca²⁺ dysregulation and arrhythmogenesis

All the mentioned modifications that can happen in the context of mitochondrial Ca²⁺

overload lead up to a Ca²⁺ dysregulation and arrhythmogenesis. First, due to sarcKATP channels opening from a low ATP/ADP ratio turns the cardiomyocyte to a hyperpolarized state. To overcome this, a stronger stimulation is needed to cross the cells threshold for a depolarization and subsequent activation of L-type channels. Second, due to lower ATP concentrations, Mg²⁺ binds to the RyR in the cytosolic side, inhibiting its opening upon stimulation. Not only that, an oxidized RyR that is subsequently phosphorylated by an oxidized CAMKII is very much likely to present with stochastic opening due to the lower luminal Ca²⁺ threshold and by its unbinding by its modulating protein calstabin2, which has been associated with development of AF in both animal models and patients (Vest et al., 2005). This manifests as a Ca²⁺ spark and, if its big enough, as a Ca²⁺ wave in the cardiomyocyte’s transient with subsequent generation of Delayed afterdepolarizations

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(DADs), events which are arrhythmogenic substrates as they are unsolicited contractions that can propagate to neighboring cardiomyocytes. It is worth noting that under these circumstances the RyR also losses its capacity to sense luminal Ca²⁺ and enter a

refractory state. This loss of a refractory state maintains the RyR open for longer periods of time, lengthening the AP. A longer AP have been associated with a longer QT interval in the electrocardiogram, an anomaly that is also associated with an increased risk of arrhythmias. Third, the SR Ca²⁺ content released is unable to be pumped back completely as SERCA has less ATP to work with, coupled with possible oxidation that reduces its functionality and a less phosphorylated PLB that binds SERCA and inhibits its activity.

This increases diastolic Ca²⁺ concentration, which further activates CAMKII which continues to phosphorylate RyR. CAMKII also phosphorylates L-type Ca²⁺ channel and NCX. A phosphorylated and gluthationylized L-type Ca²⁺ channel introduces more Ca²⁺

when an AP does pass through. To compensate the higher diastolic Ca²⁺ concentration, NCX increases the percentage of Ca²⁺ extruded to the extracellular space. This happens at the expense of slowly depolarizing the cell, as it introduces 3Na⁺ for every Ca²⁺. A lower SR Ca²⁺ content also means that with each AP, contractility will decrease as the Ca²⁺

released is much less and throughout a larger timeframe. All these changes make it more likely that the cardiomyocyte will contract stochastically and not when an appropriate AP arrives from the sinus pacemaker. Mitochondrial Ca²⁺ transport is also affected as a higher diastolic Ca²⁺ is coupled with a mCU that has a higher sensitivity due to

glutathionylation. Further mitochondrial Ca²⁺ transport creates a positive feedback loop that continues to aggravate mitochondrial dysfunction and ROS production. This cycle will eventually lead to an opening of the mPTP, which can be seen as an irreversible loss of mitochondrial function which can potentially lead to cell death. With time,

cardiomyocytes are replaced with fibrotic tissue, which generates a physical barrier for proper stimulation of the myocardium in an orderly manner. This creates possible paths for reentry arrhythmias. A summary the presented mechanisms of arrhythmias are shown in Figure 2.

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27 Figure 2: Mitochondria-SR-T tubule communication in arrhythmogenesis. Conditions which favor Ca²⁺ overload, such as chronic adrenergic stimulation causes; 1) an increased Ca²⁺ transport through mCU to the mitochondrial matrix, shortening mitochondrial Ca²⁺ transient’s amplitude and stabilizing

mitochondrial Ca²⁺ concentration at a high level which causes mitochondrial Ca²⁺ overload; 2) Increased ROS production by mitochondrial structures such as the ETC with subsequent 3) loss of ΔΨm and 4) decreased ATP production; 5) Opening of mPTP which initializes cell death signal; 6) Increased mCU activity by oxidation and possibly by AMPK phosphorylation following a decline in ATP concentration, which increases Ca²⁺ transport with more ROS production; 7) Decreased SERCA activity by oxidation and decreased PLB phosphorylation which 8) increases diastolic Ca²⁺ concentration and 9) decreases SR Ca²⁺

content, which reduces contractility; 10) Increased CAMKII activity by Ca²⁺ activation, oxidation and autophosphorylation; 11) Sensibilization of the RyR to luminal Ca²⁺content with a reduced refractory state by oxidation and CAMKII hyperphosphorylation, with a subsequent unbinding of its modulating protein, calstabin2, and less sensitized to cytosolic Ca²⁺ by Mg²⁺ binding; 12) Oxidation of L-type Ca²⁺ channel that increases the amount of Ca²⁺ that enters the cardiomyocyte upon activation; 13) Opening of sarcKATP channels by a reduced ATP/ADP ratio, which decreases the cardiomyocyte’s bathmotropism and dromotropism; 14) Increased activity of the NCX which slowly depolarizes the cardiomyocyte, which increases the risk of an unsolicited AP. All these changes combined produce; at a cellular level, Ca²⁺

mishandling with subsequent low contractility and spontaneous contraction that can propagate to neighboring cardiomyocytes and; at a tissular level, patches of slow conduction by reduced celullar dromotropism or collagen deposition caused by cardiomyocyte cell death that enable possible reentry zones for sustained arrhythmias.

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Possible new targets in SR- mitochondria interconnection and potential anti- arrhythmic drugs

Development of new therapeutics that can break the vicious cycle of Ca²⁺ overload, mitochondrial dysfunction and ROS generation may be what is needed to make a substantial improvement in reducing the incidence of sudden cardiac death and disease progression, especially in high risk patients such those with HF. Fortunately, there are a few candidates which may prove useful in doing so.

mCU inhibition by Ru360

Given that the central piece to the positive feedback cycle seems to be the mCU. mCU inhibition may be efficient in stopping the cycle and maintaining mitochondrial integrity.

Ru360 is a potent mCU inhibitor. It is an oxygen-bridged small molecule with a dinuclear ruthenium center complexed with amine groups. This molecule was first fully described in 1998, synthetizing it from a familiar component, Ruthenium Red. Ruthenium Red was first used as a mCU inhibitor to study mitochondrial Ca²⁺ dynamics but was found to be non-specific for the mCU as it had various effects on other cellular components. Ru360

was then described as being more potent (IC50 of 0.184nM vs 6.85nM for Ruthenium Red) and have a much greater affinity for the mCU, with a Kd of about 0.34nM. Doses of up to 10µM had no effect on SERCA, the RyR, NCX, L-type Ca²⁺ channels or

myofibrils. This demonstrates the high specificity for the mCU and so is safe to use without much affection in other cellular components. Since then, Ru360 has been used to research the effects of inhibiting mitochondrial Ca²⁺ transport on various in models of cardiac injury. It has already proved its efficacy in preventing early afterdepolarizations (EADs) in ventricular myocytes in a non-ischemic HF model (A. Xie et al., 2018). It has also been shown to reduce ventricular fibrillation in mice with thoracic aorta banding when presented with a catecholamine challenge. It is worth mentioning that inhibiting mitochondrial Ca²⁺ transport was related to a less oxidized RyR in isolated

cardiomyocytes as opposed to other drugs that increased mitochondrial Ca²⁺ content such as kaempferol, an mCU agonist, and CGP-37157, an inhibitor of mNCX, which enhanced RyR oxidation. Isolated cardiomyocytes also presented with improved Ca²⁺ homeostasis and less spontaneous Ca²⁺waves (Hamilton et al., 2018). This further proves that

mitochondrial Ca²⁺ overload and ROS generation can affect SR components and should be taken more into consideration. Lastly, mitochondrial Ca²⁺ transport inhibition was described as useful in maintaining a proper ECG signaling in a murine pressure overload HF model by enhancing mitochondrial autophagy (Yu et al., 2018). It has been

previously described that mitochondrial dynamics (fusion, fission and mitophagy) are impaired in a wide range of cardiovascular diseases and this could be another potential therapeutic target (Vásquez-Trincado et al., 2016). Not surprisingly, mitophagy was found to be impaired in the HF model and inhibiting mitochondrial Ca²⁺ transport seemed to avoid dysfunctional mitochondria accumulation though no clear explanation was given as to why it happened. It appears that there is much more to be described regarding the affected mitochondrial signaling mechanisms when mitochondrial Ca²⁺ overload ensues.

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Gene therapy for mCU and SERCA expression modulation

Similar to the approach of using a specific mCU inhibitor. Using gene therapy to downregulate the expression of mCU is another feasible alternative. With the development of better protocols to direct gene therapy to specific tissues, it may be possible to knockdown the expression of mCU using siRNA or other molecules.

Transgenic mice with mCU KO in cardiac tissue not only has shown to have less mPTP opening than its WT counterpart but also proved that even a complete KO of mCU is still compatible with life if not done during embryo development, as it could be inferred that without mitochondrial Ca²⁺ transport ATP production would not meet the organs

demand. KO mice appeared without any problems when resting and upon being put on a treadmill, they could even sprint for as long as their WT counterparts, provided that they were given a “warm up” of 30 minutes before hand as they were unable to do it acutely (Kwong et al., 2015). This correlates with finding similar mitochondrial basal Ca²⁺ levels between groups but needing more time to accumulate Ca²⁺ after stress stimulation in the KO group. Cardiomyocytes from the KO group also showed less mPTP opening

compared to the WT group and hearts had a less affected area when imposed with ischemic/reperfusion injury. Another study using knockdown mice for mCU in cardiac tissue showed that upon being submitted to a non-ischemic HF model they presented with reduced Na⁺/Ca²⁺ exchange currents, decreased AP duration, no EADs and reduced incidence of ventricular fibrillation compared to the WT group (A. Xie et al., 2018).

Combining these findings with the recent development of nanometric particles used for drug delivery places this type of therapy as a possible near future as it has been recently proven that siRNA can be packaged and protected from degradation while still

maintaining biological effects in in vitro studies.

Numerous studies have shown that SERCA’s functionality is reduced in common cardiac pathologies and conditions which are prone to develop arrhythmias. Such is the case of AF, in which reduced SERCA expression has been described in peripheral blood cells of patients with AF and these same levels of expression can be used to predict clinical response to treatment such as epicardial thoracoscopic pulmonary vein isolation (Sardu et al., 2020). It is no surprise that increasing SERCA’s capacity to pump Ca²⁺ into the SR to increase SR Ca²⁺ content, which in turn increases the Ca²⁺ released during CICR and translates into more contractile force generated along with a lower probability of

stochastic RyR opening by decreasing cytosolic Ca²⁺ concentration during diastole is one of the most studied approaches to battle cardiac dysfunction and one that has been

assessed from different viewpoints. Using genetic therapy is one of them. Perhaps the best example that gene therapy is a plausible treatment for cardiovascular diseases is the CUPID trials. During the first trial, a small group of patients with advanced HF received an intracoronary infusion of an adeno-associated viral vector (AAV) that contained genetic material that coded for SER-CA2a. Phase 1 of the trial was composed of only 9 patients which were followed-up for 6-12 months. These patients presented with improvements in symptomatic (NYHA and Minnesota Living with Heart Failure Questionnaire), functional (6-minute walk test and VO2 max), biomarker (NT-ProBNP)

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and LV functional parameters (EF and end-systolic volume) along with no significant changes blood chemistries, electrolytes nor kidney or liver function test, as well as reported adverse effects in other organs (Jaski et al., 2009). It was noted as well that 2 patients who did not had clinical improvement despite the therapy already had circulating antibodies against the viral vector before transfusion. For the phase 2a trial, 39 patients received intracoronary infusion of the AAV. Again, the same parameter in symptomatic, functional, biomarker and LV functional domains were assessed. The treatment group presented with improvement in the above-mentioned parameters as well as presenting with an increased time from therapy to the occurrence of clinical events related to HF and a decreased frequency of cardiovascular events in the 12 months of follow-up (Jessup et al., 2011). Given the promising results from the first clinical trials, a randomized, multinational, double-blind, placebo-controlled, phase 2b trial was conducted. 250 HF patients with reduced EF were enrolled for the study. The primary endpoint was time to recurring events, defined as either hospital admission for HF or ambulatory treatment because of worsening heart failure. Unfortunately, after a median follow-up of 17.5 months the treatment group did not improved time to recurrent events nor mortality when compared to placebo (Greenberg et al., 2016). The reason as to why the results were so different from the previous studies remain unclear. It is hypothesized that it may have been that the previous studies had results affected by chance, give the small number of patients, or that the formulation used in the CUPID 2 trial was not the most effective as there was discrepancy to what proportion of loaded particles to empty particles was the best to use to prevent neutralization while maintaining a biological effect. Even though the outcome was not the expected, it at least proved gene therapy as a viable option as there was no increase in major adverse effects when compared to the placebo group.

Furthermore, the trial provides insight as to what needs further development before a second attempt to implement gene therapy is done.

Another strategy to enhance SERCA’s Ca²⁺ pumping capacity is to functionally reduces its inhibitor protein, PLB. This strategy has been studied with a PLB KO murine model.

This model presented with a similar magnitude in the L-type Ca²⁺ channel current when compared to the wild type although with a faster decay, a larger AP that decayed faster, a greater SR Ca²⁺ content, a better Excitation-Contraction coupling (measured as Δ

cytosolic Ca²⁺ concentration/L-type Ca²⁺ channel current) and more frequent and greater Ca²⁺ sparks, although decay time was similar (Santana, Kranias, & Lederer, 1997). This model also presented with a better diastolic function in doppler and color M-mode echocardiography (Schmidt et al., 2002). More than that, combining a SERCA

overexpression model with a PLB KO model resulted in an even more enhanced cardiac state. Mice with both genetic modifications presented with higher maximal rates of contraction, maximal rates of relaxation and lower time of Ca²⁺transient decay when compared against the groups with either single mutation. No histological or pathological changes were found in the double transgenic model (W. Zhao et al., 2003). This

demonstrates the synergy achieved when enhancing SERCA’s functionality from two different approaches, making it possible address SERCA’s dysfunction with different treatments at the same time.

Instituto Tecnológico y de Estudios Superiores de Monterrey