Redox-Active Drug, MnTE-2-PyP5+, Prevents and Treats Cardiac Arrhythmias Preserving Heart Contractile Function

Background Cardiomyopathies remain among the leading causes of death worldwide, despite all efforts and important advances in the development of cardiovascular therapeutics, demonstrating the need for new solutions. Herein, we describe the effects of the redox-active therapeutic Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, AEOL10113, BMX-010 (MnTE-2-PyP5+), on rat heart as an entry to new strategies to circumvent cardiomyopathies. Methods Wistar rats weighing 250-300 g were used in both in vitro and in vivo experiments, to analyze intracellular Ca2+ dynamics, L-type Ca2+ currents, Ca2+ spark frequency, intracellular reactive oxygen species (ROS) levels, and cardiomyocyte and cardiac contractility, in control and MnTE-2-PyP5+-treated cells, hearts, or animals. Cells and hearts were treated with 20 μM MnTE-2-PyP5+ and animals with 1 mg/kg, i.p. daily. Additionally, we performed electrocardiographic and echocardiographic analysis. Results Using isolated rat cardiomyocytes, we observed that MnTE-2-PyP5+ reduced intracellular Ca2+ transient amplitude, without altering cell contractility. Whereas MnTE-2-PyP5+ did not alter basal ROS levels, it was efficient in modulating cardiomyocyte redox state under stress conditions; MnTE-2-PyP5+ reduced Ca2+ spark frequency and increased sarcoplasmic reticulum (SR) Ca2+ load. Accordingly, analysis of isolated perfused rat hearts showed that MnTE-2-PyP5+ preserves cardiac function, increases SR Ca2+ load, and reduces arrhythmia index, indicating an antiarrhythmic effect. In vivo experiments showed that MnTE-2-PyP5+ treatment increased Ca2+ transient, preserved cardiac ejection fraction, and reduced arrhythmia index and duration. MnTE-2-PyP5+ was effective both to prevent and to treat cardiac arrhythmias. Conclusion MnTE-2-PyP5+ prevents and treats cardiac arrhythmias in rats. In contrast to most antiarrhythmic drugs, MnTE-2-PyP5+ preserves cardiac contractile function, arising, thus, as a prospective therapeutic for improvement of cardiac arrhythmia treatment.


Introduction
Therapeutic improvements, lifestyle modifications, and wider adoption of evidence-based medicine have resulted in a remarkable 30-35% decline in cardiovascular mortality [1]. However, despite all efforts and advances in developing cardiovascular therapeutics, cardiomyopathies are still a major public health problem and the main causes of death around the world [2,3].
Since the first observation that Ca 2+ was required for cardiac contraction and pacemaker activity, the role of Ca 2+ as a signaling ion in the heart has been progressively dissected and better understood at molecular level, and it is clear that abnormalities in Ca 2+ homeostasis play a pivotal role in the pathogenesis of many cardiovascular diseases, including cardiac arrhythmias [4]. Inherited gene alteration and acquired defects of multiple Ca 2+ -handling proteins can contribute to the pathogenesis of arrhythmias in different categories of heart disease [4]. However, drug therapy is available only for some of these conditions and is often only partially effective [5].
Ca 2+ handling within cardiomyocytes is widely recognized as a potential target for the treatment of cardiac disease. Whereas the role of Ca 2+ channels in cardiac muscle contraction has long been elucidated [6], the biophysical and genetic identities of various voltage-gated Ca 2+ channels were disclosed [7,8], contributing along the way to several classes of antagonists being described, which comprise now part of the formulary for the treatment of cardiac diseases including arrhythmias [4].
Accordingly, Ca 2+ channel blockers are able to decrease the automaticity of ectopic foci in the heart and can be used in many arrhythmias [4]. Overall, it is thought that reduced L-type calcium currents (I Ca,L ) result in less Ca 2+ overload on the myocyte, reducing tendency to ectopy, which can trigger arrhythmias [4]. Additionally, cardiotonic glycosides or digitalis are positive inotropes used in clinical practice for the treatment of heart failure that also behave as endogenous ligands for Na + /K + ATPase. An increase in intracellular Ca 2+ content mediates their positive inotropic effect but has also been suggested as a trigger of life-threatening arrhythmias [9].
In many tissues, including the heart, reactive oxygen/nitrogen species (ROS/RNS) are often derived from mitochondria, NADPH oxidase, or uncoupled-nitric oxide synthase (NOS) and are kept under tight homeostatic control [10,11]. In the cardiovascular system, ROS/RNS has been shown to play an important role in regulation of K + , Na + , L-type Ca 2+ channels (in plasmatic membrane), and ryanodine receptor (RyR2) in sarcoplasmic reticulum membrane [12][13][14]. Mn-porphyrin-based compounds have been widely recognized as potent redox-active therapeutics, being able to modulate ROS/RNS in several animal models of oxidative stress [15][16][17][18]. Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP 5+ ), also known as AEOL10113 or BMX-010, is currently under phase I/II clinical trials in Canada and the USA [15], and preclinical toxicological studies in conscious telemetered male cynomolgus monkeys showed that administration of MnTE-2-PyP 5+ at a dose of 1 mg/kg/day led to no statistically significant changes in heart rate or arterial blood pressure [19].
The pharmacokinetic studies on MnTE-2-PyP 5+ show a good distribution of this compound into the heart [20,21]. Such data prompted us to investigate MnTE-2-PyP 5+ as a redox-active experimental therapeutic for cardiomyopathy, with particular focus on reducing Ca 2+ stress and preserving cardiac contractile function. We demonstrate that MnTE-2-PyP 5+ exerts protective effects in rat hearts, by modulating Ca 2+ dynamics, reducing arrhythmia score, and preserving contractile function of the heart. Additionally, MnTE-2-PyP 5+ was effective in preventing and treating cardiac arrhythmias in vivo.

Methods
2.1. Animals. All experiments were performed using rats of both sexes (Rattus norvegicus, 200-250 g). Animals were maintained at the Federal University of Paraiba (UFPB), Brazil, in accordance with NIH guidelines for the care and use of animals. Experiments were performed according to approved animal protocols from the Institutional Animal Care and Use Committee at UFPB (CEUA Protocol 016/2017). All animals were euthanized by decapitation.
2.3. Cardiomyocyte Isolation and Ca 2+ Recordings. Ventricular rat cardiomyocytes were isolated and stored until they were used as previously described [30]. Intracellular Ca 2+ analysis was performed with Fluo-4 AM (10 μM; Invitrogen, Eugene, OR)-loaded cardiomyocytes. The cells were stained for 30 min and then washed to remove the excess dye. Cells were electrically stimulated at 1 Hz to produce steady-state conditions. The images were recorded in a Zeiss LSM 510META confocal microscope. As an indicator of the SR Ca 2+ load, 10 mM caffeine stimulation (in a Ca 2+ -and Na +free solution) and the amplitude of the Ca 2+ transient evoked were recorded [31]. Preconditioning pulses (1 Hz) were used in the cells before caffeine was applied. Ca 2+ spark frequencies were recorded in resting ventricular myocytes. The Ca 2+ level was reported as F/F 0 (or as ΔF/F 0 ), where F 0 is the resting Ca 2+ fluorescence.

ROS Recordings.
Isolated cardiomyocytes were incubated with 10 μM dihydroethidium (DHE, Molecular Probes, Eugene, OR) for 30 min at 37°C and were subsequently washed with an extracellular solution to remove the excess dye. Images were acquired with a Zeiss LSM 510 META confocal microscope. Images were analyzed in ImageJ software.
2.6. Measurement of LV Myocyte Shortening. Cellular contractility was evaluated as previously described [34]. Briefly, isolated myocytes were placed in a chamber mounted on the stage of an inverted microscope (Eclipse TS 100; Nikon, Japan 2.10. In Vitro Arrhythmia. In vitro arrhythmia was determined in an isolated heart as described previously [35]. Hearts were subjected to perfusion with KHS containing 1.25 mM of calcium (control group) at 34°C during 20 min. After stabilization, the hearts were subjected to perfusion with 3.3 mM high calcium (HC group) or with high calcium in the presence of 20 μM MnTE-2-PyP 5+ during 25 min. Arrhythmia scores were determinate as previously described [36]. Therefore, to quantify the arrhythmias, 25 min of experiment was divided into 3 min intervals and the arrhythmia scores were added at the end.  (2-200 μM), and observed a significant reduction in peak Ca 2+ transient in a concentration-dependent manner (Figure 1(a)). However, the kinetics of Ca 2+ decay was altered only for T90 at 200 μM concentration (Figures 1(b)  Considering that MnTE-2-PyP 5+ is a superoxide dismutase (SOD) mimetic (currently recognized as a broad redox modulator) [17,18], we used dihydroethidium (DHE), a fluorescent, cell-permeable, reactive oxygen species (ROS) indicator, to evaluate MnTE-2-PyP 5+ effect on cardiomyocyte ROS levels. MnTE-2-PyP 5+ did not change the basal levels of ROS. However, as its well-known that MnTE-2-PyP 5+ , physiologically, has antioxidant activity under oxidative stress conditions, we used isoproterenol (ISO) as a cell stressor. As expected, ISO induced an increase in DHE fluorescence that was prevented by MnTE-2-PyP 5+ (Figures 2(a) and 2(b)), indicating that MnTE-2-PyP 5+ is efficient in modulating cardiomyocyte redox state under stress conditions. Nevertheless, as MnTE-2-PyP 5+ did not change basal ROS levels in cardiomyocytes, this result suggests that the observed reduction in Ca 2+ transient induced by MnTE-2-PyP 5+ is likely independent of its antioxidant effect.
As we observed all these alterations in pivotal components of excitation-contraction coupling (ECC) and considering the importance of Ca 2+ ion for cellular contraction, we next analyzed the MnTE-2-PyP 5+ effects on cardiomyocyte contractility. Remarkably, despite the reduction in I Ca,L and in peak Ca 2+ transient, no changes in fractional shortening, systolic length, or diastolic length of MnTE-2-PyP 5+treated cardiomyocytes were observed, which is in direct contrast to the LTCC blocker verapamil-treated group (Figures 3(a)-3(d)). This result is noteworthy, because it indicates that although MnTE-2-PyP 5+ decreases Ca 2+ transient, it preserves cardiomyocyte contractility.
As we observed alterations in Ca 2+ spark frequency in isolated cardiomyocytes, we also tested if MnTE-2-PyP 5+ altered SR Ca 2+ content in isolated atria. Consistent with isolated cell data, MnTE-2-PyP 5+ increased the SR Ca 2+ load in approximately 66%, which helps to explain the maintenance of the cardiac contractility ( Supplementary Figs. S1D and E). 4 Oxidative Medicine and Cellular Longevity

Oxidative Medicine and Cellular Longevity
Considering the involvement of Ca 2+ ions in triggering cardiac arrhythmias, we used Langendorff-perfused hearts to test whether MnTE-2-PyP 5+ could alter the electrical activity of the heart. Electrocardiographic (ECG) recordings were used to analyze ECG intervals and segments. The results showed that heart rate and QRS complex length were not modified by MnTE-2-PyP 5+ (Figures 4(a)-4(c)). However, QTcV was significantly shortened and PRi increased (Figures 4(d) and 4(e)). Additionally, to investigate if MnTE-2-PyP 5+ could target cardiac arrhythmias, isolated  7 Oxidative Medicine and Cellular Longevity hearts were perfused with high calcium (HC) to induce cardiac arrhythmias. Figure 4(f) shows normal ECG traces in control situation (top panel) and induced arrhythmias in HC-perfused heart (bottom panel). As shown in Figure 4(g), HC perfusion significantly increased the arrhythmia scores and MnTE-2-PyP 5+ decreased HC-induced cardiac arrhythmias. Furthermore, most of the arrhythmias evidenced in control situation were of lower severity, such as ventricular premature beats (VPB, Figure 4(h)). In contrast, HC-perfused hearts presented ventricular fibrillation (VF), the most severe type of arrhythmia. Remarkably, MnTE-2-PyP 5+ prevented the incidence of the most severe arrhythmia events (Figure 4(h)).

MnTE-2-PyP 5+ Increases Ca 2+ Transient and Preserves
Heart Function In Vivo. Based on our in vitro results, we visualized MnTE-2-PyP 5+ as a potential lead for therapeutic approaches for some cardiac arrhythmias. Thus, we designed in vivo experiments in rats to investigate the effect of MnTE-2-PyP 5+ on the heart. Animals were treated daily with 1 mg/kg MnTE-2-PyP 5+ (i.p. injections) for 15 days. First, we tested MnTE-2-PyP 5+ effects on Ca 2+ transient. Cardiomyocytes from MnTE-2-PyP 5+ -treated rats presented increased peak Ca 2+ transient and T90 time for Ca 2+ decay, different from what we observed in acutely treated isolated myocytes (Figures 5(a)-5(c)). Additionally, as we verified an increase in SR load in vitro, we decided to investigate the SR load in cardiomyocytes isolated from treated animals. In agreement with in vitro experiments, MnTE-2-PyP 5+ treatment increased SR Ca 2+ load by approximately 14% (Figure 5(d)).
Additionally, heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) ratios were evaluated as markers of cardiac hypertrophy in MnTE-2-PyP 5+ -treated animals. As shown in Figures 5(e) and 5(f), these parameters did not differ from those of the control group. Next, by echocardiographic analysis, we verified that MnTE-2-PyP 5+treated animals had no difference in ejection fraction (EF) when compared to control ( Figure 5(g)), indicating that MnTE-2-PyP 5+ preserves cardiac contractility in vivo. Taken together, these results indicate that MnTE-2-PyP 5+ administered at 1 mg/kg/day i.p. does not alter normal heart function in vivo.

MnTE-2-PyP 5+ Effectively Prevents and Treats Cardiac
Arrhythmias In Vivo. Considering the robust effects of MnTE-2-PyP 5+ in preventing cardiac arrhythmias in isolated hearts, we assessed its antiarrhythmic property in vivo. By using a dexamethasone-induced arrhythmia model, we tested both preventive and therapeutic actions of MnTE-2-PyP 5+ . Figure 6(a) shows representative ECG traces along with the type of the recorded arrhythmias: isolated VPB, sustained VPB, and VT. Remarkably, in both prevention and treatment protocols, MnTE-2-PyP 5+ was completely effective to reduce both arrhythmia score and duration (Figures 6(b) and 6(c)), restoring the control profile. When we analyzed the severity of ventricular arrhythmias, we observed that MnTE-2-PyP 5+ prevented the occurrence of ventricular tachycardia (VT) (Figure 6(d)). However, although MnTE-2-PyP 5+ reduced the duration of arrhythmias, it was not able to reverse the relative occurrence of VT ( Figure 6(d)).
To better view the multiple actions of MnTE-2-PyP 5+ , Figure 7 presents a schematic summary of the main effects of MnTE-2-PyP 5+ both in vitro and in vivo.
To better view the multiple actions of MnTE-2-PyP 5+ , Figure 7 presents a schematic summary of the main effects of MnTE-2-PyP 5+ both in vitro and in vivo.

Discussion
Cardiac arrhythmias are important causes of sudden death; thus, proper treatment of these conditions is of utmost importance. Indeed, over the last two decades, there has been a great deal of progress in arrhythmia management. However, most antiarrhythmic drugs proved ineffective or dangerous in patients with ventricular arrhythmias [40], demonstrating the need for new therapeutic strategies.
Although the mainstay of treatment for catecholaminergic polymorphic ventricular tachycardia (CPVT) has been β-blockade, there has also been early evidence that blocking I Ca,L with the LTCC blocker verapamil prevents ventricular arrhythmias [41]. Overall, it is thought that reduced I Ca,L results in less Ca 2+ overload of the myocyte, reducing predisposition to ectopy that can trigger arrhythmias [4]. In agreement with these findings, our study shows that acute administration of MnTE-2-PyP 5+ to isolated cardiomyocytes reduced the peak Ca 2+ transient in these cells, in association with reduced I Ca,L . Additionally, acute administration of MnTE-2-PyP 5+ in isolated hearts resulted in reduction in arrhythmia index, severity, and duration of arrhythmias, demonstrating, for the first time, that MnTE-2-PyP 5+ represents a new lead molecule for the treatment of cardiac arrhythmias.
Additionally, while in vitro acute use of MnTE-2-PyP 5+ reduced Ca 2+ transient in cardiomyocytes, in vivo 15-day use of MnTE-2-PyP 5+ in healthy rats did not change Ca 2+ transient in cardiomyocytes. Although these results may at first seem inconsistent, when we consider that MnTE-2-PyP 5+ reduced Ca 2+ spark rate and increased SR load, chronically, these two effects combined may account for the final increased Ca 2+ transient observed in vivo. Additionally, as we and others [15,17,18] demonstrated that under stress conditions MnTE-2-PyP 5+ prevents oxidative stress, this effect can also contribute, chronically, to cellular restoration of basal transient kinetics. Accordingly, Almeida et al. [42] demonstrated that aldosterone-treated cardiomyocytes presented increased I Ca,L and Ca 2+ transient and that Angiotensin-(1-7) restored basal I Ca,L albeit with a great increase in Ca 2+ transient. Further investigation demonstrated that this alteration in Ca 2+ transient was caused by reduction in Ca 2+ spark frequency and consequent increased SR load. MnTE-2-PyP 5+ apparently works via a similar mechanism by improving Ca 2+ transient in the long term. In addition, the systemic antioxidant effect of MnTE-2-PyP 5+ must be considered in the cardiovascular health in vivo.
By reducing peripheral vasoconstriction and LV afterload, calcium channel blockers were thought to have a potential role in the management of chronic heart failure (HF). However, first-generation dihydropyridine and nondihydropyridine calcium channel blockers also have myocardial depressant activity [43]. Several clinical trials have demonstrated either no clinical benefit or even worse outcomes in patients with HF treated with these drugs [44][45][46][47][48]. Despite their greater selectivity for calcium channels in vascular smooth muscle cells, second-generation calcium channel blockers, dihydropyridine derivatives such as amlodipine and felodipine, have failed to demonstrate any functional or survival benefit in patients with HF [49][50][51][52][53]. Together, these data show that although calcium channel blockers have an important role in the management of cardiac arrhythmias, they are of limited use, especially in patients with HF.
Although the use of calcium channel blockers for arrhythmia treatment is often plagued by myocardial depressant activity, here, we demonstrated that MnTE-2-PyP 5+ prevents and treats cardiac arrhythmias while preserving contractility at both cardiomyocyte and heart levels. These combined effects respond to a large gap in arrhythmia treatments, especially in patients with HF.
It is worth noting that our study demonstrates that MnTE-2-PyP 5+ preserves cardiomyocyte and heart contractility and exerts antiarrhythmic effects both in vitro and in vivo, representing, thus, a potentially new strategy to treat cardiac arrhythmias in patients with contractile dysfunctions. In addition, although reduction in peak calcium transients is usually related to reduction in cardiac contractility, modulation of proteins involved in calcium handling or contractile machinery can alter this relationship. In this way, Vanzelli el al [54]. demonstrated that heart failure mice treated with carvedilol had an improvement in cardiac fractional shortening instead of no alterations in peak calcium transients. Although we did not analyze these mechanisms directly, we speculate that MnTE-2-PyP 5+ effect may be somewhat related to the mechanism described by Vanzelli el al [54].; further investigations are obviously

12
Oxidative Medicine and Cellular Longevity needed to disclose the actual MnTE-2-PyP 5+ mechanism(s) of action. Finally, it is important to highlight that the use of Mn porphyrin-based SOD mimics for cardiovascular treatments is in its infancy. A first report [55] was very recently published on the ability of an analogous Mn porphyrin, MnTnBuOE-2-PyP 5+ (BMX-001) [28], to suppress aortic valve sclerosis in mice and human models. We showed herein that the prototypical Mn-porphyrin MnTE-2-PyP 5+ represents a simple, promising redox-active therapeutic for preventing and treating cardiac arrhythmias, preserving heart contractile function. Taken together, the data strengthen the therapeutic potential of Mn porphyrins in a quite unexplored field of cardiac applications.

Data Availability
Part of this manuscript is under patent registration. Because of that the authors do not present a data availability statement.