Title of Invention: A Method for Treating Metabolic Cardiomyopathy Caused by A Metabolic Hepatic Diseases

Technical Field

[0001] The present disclosure describes a method for treating a plurality of cardiovascular diseases caused by a liver disease in a subject, especially in a mammal, through administration of a low dosage of a composition of a biguanide component, as well as a cyclosporine.

Background Art

[0002] Cardiovascular diseases are one of the most commonly known diseases in the world that cause death. Liver failure and liver diseases are among the factors that lead to various types of cardiovascular diseases as well as the risk of cardiovascular diseases increases in those people with these types of liver dysfunctionality.

[0003] Metabolic hepatic cardiomyopathy is one of the heart diseases that was observed in patients with a metabolic hepatic disease. The Metabolic hepatic cardiomyopathy is characterized by the impaired systolic response to physical stress, diastolic dysfunction, and electrophysiological abnormalities, especially QT interval prolongation.

[0004] On the other hand, ischemic preconditioning (IPC) is a potent protective phenomenon against ischemia-reperfusion (l/R) injuries. The most important pathways in IPC are activation of adenosine monophosphate kinase (AMPK) as well as inhibition of the onset of the mitochondrial permeability.

[0005] Furthermore, it has been demonstrated that a cardioprotective effect of the IPC in diabetic cardiomyopathy and hypercholesterolemia cardiomyopathy is abolished. Thus, by considering (i) the critical role of MPTP in necrosis and apoptosis in IPC phenomenon, (ii) the role of activation of AMPK in the regulation of the interplay between apoptosis and autophagy in diabetic cardiomyopathy (DCM), and (iii) the similarity between diabetic cardiomyopathy (DCM) and cirrhotic cardiomyopathy (CCM) respectively it seems that inhibition of autophagy and induction of mPTP via inactivation of AMPK can play an essential role in blunting the cardioprotective role of I PC in cirrhotic cardiomyopathy.

[0006] Therefore, a method for treating a heart disease caused by a liver dysfunction by using at least one component that has a suitable effect on the above- mentioned issues as well as can treat and/or prevent the heart disease is required. Thus, a method based on using a composition of a biguanide component along with a cyclosporine component is developed to improve or prevent the heart diseases caused by a liver disfunction.

Summary of Invention

[0007] This summary is intended to provide an overview of the subject matter of this patent, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. The proper scope of this patent may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0008] In a general aspect, the present disclosure is directed to an exemplary method for treating a plurality of heart diseases caused by a liver disease. The exemplary method may comprise administrating an effective dosage of a composition of biguanide component or a pharmaceutically acceptable composition thereof to a subject in need thereof.

[0009] The above general aspect may have one or more of the following features. In some exemplary implementations, the exemplary method may further comprise administrating a therapeutical effective amount of a cyclosporine component or a pharmaceutically acceptable composition thereof to the subject. In an exemplary implementation, the plurality of heart diseases may comprise acute coronary syndrome, arrhythmia, heart failure, heart metabolic enzyme disorders, myocardial infarction, myocardial fibrosis, myocardial necrosis, and ischemia. In an exemplary implementation, the composition of biguanide component or a pharmaceutically acceptable composition thereof may have the effective dosage in a range of 25 mg to 100 mg per a day. In some exemplary implementations, the cyclosporine component or a pharmaceutically acceptable composition thereof may administer to the subject in a range about 12 mg/ml to 25 mg/ml per a week. In an exemplary implementation, the subject may be a mammal. In some exemplary implementations, the subject may be a human. In an exemplary implementation, the liver disease that resulted in the heart disease may comprise liver fibrosis, a type of hepatitis, a type of fatty liver, cholestasis, or a type of liver cirrhosis.

[0010] In another general aspect, the present disclosure is directed to use of a composition of a biguanide component or a pharmaceutically acceptable composition thereof in a low effective dosage for treating a plurality of heart diseases caused by a liver disease in a mammal.

Brief Description of Drawings

[0011 ] The drawing figures only demonstrate one or more embodiments in accord with the present teaching, by way of example only, not by way of limitation. Therefore, the drawing figures do not limit the extent of the present disclosure. Also, reference numerals with similar numbers in the figures demonstrate similar or the same elements.

Fig.1

[0012] [Fig.1 ] illustrates a curve of concentration-dependent effects of metformin (0.5-2 pM) on nodal electrophysiological properties to calculate EC50, consistent with one or more exemplary embodiments of the present disclosure. The AH, ERP, FRP, and WBCL abbreviations represented Nodal conduction time, Effective refractory period, Functional refractory period, and Wenckebach cycle length, respectively.

Fig.2

[0013] [Fig.2] illustrates a plot of right-upward shift of a recovery curve in one preparation in a presence of different concentrations of Metformin, consistent with one or more exemplary embodiments of the present disclosure. A2H2 and ms means the time course of AV conduction time and milliseconds, respectively. C1 = 1 pM Metformin, C2= 2 pM Metformin.

Fig.3

[0014] [Fig.3] illustrates a reentrant circuit tachycardia as a ratio of AVERP divided by tachycardia cycle length (T) versus tachycardia rate calculated on the basis of the observed and the functional model, consistent with one or more exemplary embodiments of the present disclosure. AVERP: AV effective refractory period; C1 = 1 pM Metformin, C2= 2 pM Metformin, verapamil in a concentration of 0.1 pM; ms: milliseconds.

Fig.4

[0015] [Fig.4] illustrates a plot of H-H intervals recorded during experimental atrial fibrillation, in a presence (B) and absence (A) of Nadolol (0.1 pM), consistent with one or more exemplary embodiments of the present disclosure. C1 = 1 pM Metformin, C2= 2 pM Metformin.

Fig.5

[0016] [Fig.5] illustrates two histopathology images of cirrhosis with different magnitude 20 pm (right) and 100 pm (left), consistent with one or more exemplary embodiments of the present disclosure.

Fig.6

[0017] [Fig.6] illustrates time course changes in left ventricular developing pressure (LVDP) (%) during a stabilization and reperfusion step, consistent with one or more exemplary embodiments of the present disclosure. (A), (B), and (C) represented a comparison between time course changes in LVDP (%) of IPC and Met in a normal status and BDL rat. (D) represented a time-dependent model of LVDP (%) changes during reperfusion step in the normal statues and BDL rat. The values are calculated as a percent of baseline and are a mean±SEM, in each group n=13. The symbols indicate significance: *, #P<0.05 compared to l/R; §P<0.05 compared to l/R (BDL); ⁽ l ⁾ P<0.05 compared to A (IPC-I/R).

Fig.7

[0018] [Fig.7] illustrates time course changes in rate-pressure product (RPP) (%) during a stabilization and reperfusion step, consistent with one or more exemplary embodiments of the present disclosure. (A), (B), and (C) represented a comparison between time course changes in RPP (%) of IPC and Met in a normal statue and BDL rat. (D) represented a time-dependent model of RPP (%) changes during reperfusion step in the normal statues and BDL rat. The values are calculated as a percent of baseline and are a mean±SEM, in each group n=7. The symbols indicate significance: *, #P<0.05 compared to l/R; §P<0.05 compared to l/R (BDL); ⁽ l ⁾ P<0.05 compared to A (IPC-I/R). Fig.8

[0019] [Fig.8] illustrates the effects of l/R, IPC, BDL, IPC (BDL), Metformin (Met), Metformin (BDL), Cyclosporine A, and Cyclosporine A (BDL) groups on a myocardial area at risk (AAR/LV%) and an infarct size (IS/AAR%), consistent with one or more exemplary embodiments of the present disclosure. Data are presented as mean ± SEM. *P<0.05 vs. IR group, §P<0.05 vs. I/R (BDL) group.

Fig.9

[0020] [Fig.9] illustrates the effects of l/R, IPC, BDL, IPC (BDL), Metformin (Met), Metformin (BDL), Cyclosporine A, and Cyclosporine A (BDL) groups on a arrhythmia score during 30 minutes ischemia and 120 minutes reperfusion, consistent with one or more exemplary embodiments of the present disclosure. Data are presented as mean ± SEM. *P<0.05 vs. IR group, §P<0.05 vs. l/R (BDL) group.

Description of Embodiments

[0021] In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well-known methods, procedures, and/or components have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present disclosure is defined only by the appended claims.

[0022] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be readily apparent to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0023] The present disclosure describes an exemplary method for treating a plurality of heart diseases caused by a liver disease. The exemplary method may comprise at least one of two administration steps. A first step may comprise administrating an effective dosage of a biguanide component or a pharmaceutically acceptable composition thereof to a subject in need thereof. A second step may comprise administrating a cyclosporine component or a pharmaceutically acceptable composition thereof at a therapeutical effective amount to the subject. Some benefits from utilizing the exemplary method described within the present disclosure may include, but are not limited to, preventing of the heart diseases such as cardiomyopathy from various metabolic disorders such as diabetic, hyper-lipoproteonemia, cirrhosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) in a low dosage of the biguanide component or the pharmaceutically acceptable composition thereof and/or the cyclosporine component or the pharmaceutically acceptable composition thereof. The biguanide component can prevent a conversion of myofibroblasts to fibrous and necrotic tissue by acting on the Janus kinase/signal transducer and activator of transcription (JAK/STAT), AMPK signaling pathway by preventing the conversion of fibroblasts to myofibroblasts.

[0024] In an exemplary embodiment, the terms “treating” and/or “treatment” may refer to prevent or remedy a plurality of heart diseases and/or improve a condition related to the plurality of heart disease that may be cause by a liver disease in a subject.

[0025] In an exemplary embodiment, the term an “effective dosage” may refer to a low amount of the biguanide component and/or the pharmaceutically acceptable composition thereof in a range of 25 mg to 100 mg per day that may be administrated to the subject at intervals of 1 to 8 weeks. [0026] In an exemplary embodiment, the terms “administrated”, “administrating” and/or “administer” may refer to administration of a pharmaceutical composition (e.g., a cyclosporine component and/or a biguanide component) to achieve a therapeutic propose (e.g., heart diseases caused by a liver disease).

[0027] In some exemplary embodiments, an exemplary method may comprise administrating a cyclosporine component or a pharmaceutically acceptable composition thereof in a therapeutical effective amount to the subject. In an exemplary embodiment, the expression a “pharmaceutically acceptable composition” may refer to a composition of the cyclosporine component that may have an effect on a mitochondrial permeability transition pore (MPTP) channel.

[0028] In some exemplary embodiments, the expression a “therapeutically effective amount” may refer to an amount in a range of 12 mg/ml to 25 mg/ml per day that may be administrated to the subject at intervals of 1 to 8 weeks.

[0029] In some exemplary embodiments, the expression a “therapeutically effective amount” may refer to an amount in a range of 12 mg/ml to 25 mg/ml per week that may be administrated to the subject at intervals of 1 to 8 weeks.

[0030] In some exemplary embodiments, the expression a “therapeutically effective amount” may refer to an amount in a range of 12 mg/ml to 25 mg/ml per day that may be administrated to the subject for the first week and an amount in a range of 12 mg/ml to 25 mg/ml per week that may be administrated to the subject at intervals of the second week to the eighth week.

[0031] In an exemplary embodiment, the term “subject” may refer to all humans and non-human animals. In some exemplary embodiments, the term “non-human animals” may refer to mammals such as, but are not limited to, non-human primates, cats, dogs, sheep, rodents, rabbits, or ferrets. In a preferred embodiment, the subject may be a human being. The terms “subject” and “patient” may be used interchangeably.

[0032] In an exemplary embodiment, aspects and features of an exemplary heart disease treatment method based on administration of at least one of a biguanide component and/or a cyclosporine component will be described in greater detail, below. TREATMENT OF A HEARS DISEASE CAUSED BY A LIVER DISEASE THROUGH ADMINESTRATION OF A BIGUANIDE COMPONENT AND A CYCLOSPORINE COMPONENT

[0033] Mitochondria play a very important role in cell death and life. In healthy myocytes, one of the primary functions of mitochondria is to produce adenosine triphosphate (ATP) that is energy-carrying molecule through oxidative phosphorylation. Mitochondria may be a pathology site for a heart damage caused by liver metabolic disease, which leads to a large increase in production of reactive oxygen species (ROS) in mitochondria by altering the balance of ROS production. Therefore, the increment of ROS may lead to reduction of autophagy and raising apoptosis due to diminishment mitochondrial kinases activities. Furthermore, through the adenosine monophosphate kinase (AMPK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways, the conversion of the fibroblast to the myofibroblast increases and resulting in expansion of necrosis and fibrosis in the heart cells.

[0034] In an exemplary embodiment, an exemplary heart disease treatment method may comprise administrating a biguanide component or a pharmaceutically acceptable composition thereof to a subject in need thereof. The administration of the biguanide component or the pharmaceutically acceptable composition thereof may cause increasing amp-kinases activities of mitochondria as well as decreasing apoptosis and increment of autophagy. In addition, the administrating the biguanide component or the pharmaceutically acceptable composition thereof may reprogram the myofibroblast cells phenotype to the fibroblast cells phenotype through the JAK/STAT signaling pathway and resulted in preventing or treatment of a myocardiopathy disease.

[0035] In an exemplary embodiment, the biguanide component or the pharmaceutically acceptable composition thereof may comprise, but are not limited to, Metformin, Imeglimin, or a combination thereof.

[0036] In an exemplary embodiment, the biguanide component or the pharmaceutically acceptable composition thereof may administrate to a patient in an effective dosage. In an exemplary embodiment, the effective dosage may be adjusted in a range of 25 mg to 100 mg that orally administrated to the patient every day. In an exemplary embodiment, the administration of the biguanide or the pharmaceutically acceptable composition thereof may be performed for 2 months.

[0037] In an exemplary embodiment, the exemplary heart disease treatment method may further comprise administrating a cyclosporine component or a pharmaceutically acceptable composition thereof to the subject. In an exemplary embodiment, the administration of the cyclosporin component or the pharmaceutically acceptable composition may block the mitochondrial permeability transition pore resulting in reprograming the myofibroblast cells phenotype to the fibroblast cells phenotype. Therefore, it affects a heart disease condition and may prevent or treat a heart disease.

[0038] In an exemplary embodiment, the cyclosporine component or the pharmaceutically acceptable composition thereof may administrate to a patient in a therapeutically effective amount such that the therapeutically effective amount may be adjusted in a range of 12 mg/ml to 25 mg/ml. In some exemplary embodiment, the cyclosporine component or the pharmaceutically acceptable composition thereof may be administrated to the patient every week. In some exemplary embodiments, the therapeutically effective amount of cyclosporine component or the pharmaceutically acceptable composition thereof may be administrated to the patient every day for 1 week and may be weekly administrated in intervals of 2 weeks to 8 weeks.

[0039] In an exemplary embodiment, the cyclosporine component or the pharmaceutically acceptable composition thereof may be administrated to the subject in a liposomal form. The liposomal form may increase the bioavailability of the cyclosporine component or the pharmaceutically acceptable composition thereof resulting in increment reabsorption of the cyclosporine component or the pharmaceutically acceptable composition thereof in the heart cells.

[0040] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Examples

[0041 ] Example 1 : Anti-AF Effects of Metformin in Mathematical Experimental Model of Arrhythmia in Isolated Rabbit Atrioventricular (AV)Node

[0042] In Example 1 , anti-AF effects of metformin in mathematical experimental model of arrhythmia in isolated rabbit AV node was investigated, consistent with the teachings of the exemplary embodiments of the present disclosure.

Induction of experimental Atrioventricular nodal reentry tachycardia (AVNRT) and Atrial fibrillation (AF)

[0043] An experimental AVNRT model was simulated in a separate series of 7 rabbit hearts, as previously described by Khori et al., (khori.et al, “Acute effects of simvastatin to terminate fast reentrant tachycardia through increasing wavelength of atrioventricular nodal reentrant tachycardia circuit. Fundamental & clinical, pharmacology”, 29(1 ), 41 -53(2015), which is hereby incorporated by reference). The experiments were designed based on the control vs. metformin response. Different concentrations of Metformin were added to the circulation so that the tissue was exposed to the metformin for 40 minutes. The simulated nodal arrhythmia was applied before and after addition of the metformin (N=7).

[0044] A slow and fast pathway was detected by forward and backward stimulation by an electrode. In fact, a stimulus was delivered by an electrode located in the upper part of the intera-Aterial septum (upper atrium) with a predefined HA interval delay. An experimental AVNRT can be considered a recurrent arrhythmia that has two forward and backward paths and when a signal is routed from a slow path forward, it completes the recirculation cycle from a fast path. In thedescribed model, the activation of the hiss handle is measured by the electrode and then completes the electrical stimulation of the rapid recirculation arrhythmia path.

The electronic component of the circuit therefore, an electrical branch of the circuit mimics a lateral path between the ventricles and the atria. The stimulation was then repeated with the His-Stimulus interval selected and the cycle recurred repeatedly. Each induction of AVNRT was followed by a recovery period of at least 10 min. A wide range of HA intervals (40-300 ms) was applied. The anti- AVNRT pleiotropic direct effects of metformin to suppress AVNRT depends on a balance between two intrinsic nodal factors: a trend to increase the AVERP, which makes tachycardia less likely, and a trend to slow the tachycardia by slowing AV nodal conduction.

[0045] The result of these opposing properties can be determined by applying the concept of wavelength, a wavelength of a reentrant circuit (A) is equal to a product of average conduction velocity (CV) and a longest refractory period (RP) and was characterized by equation (1 ):

A= CV x RP (1).

[0046] The wavelength must be shorter than the reentrant circuit (L) if a reentry is to be sustained. The Mean CV is given by a length of the reentrant circuit divided by a revolution time of the tachycardia according to equation (2):

CV =L/T (2)

[0047] During sustained tachycardias, T equals the tachycardia cycle length. Substituting Eq (2) for Eq (1 ) and rearranging the terms resulted in equation (3):

A/L=AVERP/T (3)

[0048] AVERP/T increases along with the frequency of stimulations. As the rate increases, this ratio exceeds unity, and the tachycardia cannot be sustained and is terminated. By increasing A slope, Anti-AVNRT pleiotropic effects of metformin would be expected to terminate sustained AVNRT. According to AVNRT model, metformin terminate tachycardia by increasing the index of AVERP/ tachycardia cycle length

[0049] Anti-AF effect of metformin: in the experimental setting we executed the simulated random high-rate AF by computer in 75-125-ms coupling intervals. In each AF protocol, 1500 beats over 5 minutes were stimulated and the mean interval, the shortest interval, the longest H-H interval, and the concealed beats were recorded. Briefly, the zone of concealment (ZOC) was determined by considering a time interval between a nodal and atrial ERP in a nodal recovery curve; then, the concealed conduction plotted the curve by interpolating a concealed beat before each test beat. A rate-dependent impact of the ZOC was measured by applying different stimulation cycle lengths (300-160 ms). The ZOC was calculated as a difference between the atrial refractoriness (AERP) and the nodal refractoriness (NERP). In addition, the differences between the AH and ERP were measured as an excitability index, the anti-AF effects of metformin (0.5-2pM) was calculated in control and after running aforementioned protocol.

Statistical analysis

[0050] The results were reported as the Mean SEM., and comparisons among multiple groups were made by a two-way analysis of variance. The comparisons between the two groups were made with a paired T test. A probability of 5% was taken to indicate statistical significance. Non- linear curve fitting was performed with Marquard’s technique.

Results

[0051] Fig.1 illustrates a concentration-dependent effect of metformin (0.5-2 pM) on nodal electrophysiological properties to calculate EC50. As illustrated in Fig.1 , metformin does not have significant changes in the effective refractory period (ERP). was not significant at the concentrations of 0- 2 pM of metformin. On the other hand, metformin caused a significant increase in a functional refractory Period (FRP). Furthermore, the WBCL was measured as an indicator of the refractoriness and was increased in both concentrations of 15 and 30 mg/L compared to other concentrations p<0.05).

[0052] Fig.2 shows a plot of right-upward shift of a recovery curve in one preparation in a presence of different concentrations of Metformin. 1 and2 pM concentration of Metformin significantly increased the number of concealed AV beats. In addition, an average of two subsequent records from His bundle (H-H) was increased significantly in both concentrations (p<0.05) (Fig.2).

Effects of Metformin on a AV reentrant tachycardia

[0053] As illustrated in Fig.3, Metformin suppressed AVNRT by a frequencydependent increase in A. Pronounced Metformin effects were observed in fast tachycardia cycle lengths. Metformin increased the A slope, led to greater values of AVERP/T for tachycardias of equal rate and tachycardia termination at the slower rates (Fig.3).

[0054] There is good agreement between the model calculations and experimentally observed data points for Metformin and verapamil (Fig.3). Under control conditions, AVERP/T increased as tachycardia rate increased. The model output and observed data are qualitatively superimposed in both the control and Metformin. AVERP/T is prolonged by Metformin (2 pM) to a substantially greater extent than by Metformin (1 pM) as the AVNRT cycle length decreases (Fig.3).

[0055] As well as, Fig.4 shows a goodness of a model in predicting a relationship between AVERP divided by a tachycardia cycle length during various AVNRTs. Effect of Nadolol on Metformin -induced simulated model of atrial fibrillation: Nadolol inhibited Metformin -induced depressant effect and the mean value of H- H interval. The mean number of canceled beats increased, but was not significant (Fig 4).

[0056] As a result, Metformin has a cardio protective effect against hepatic cardiomyopathy which is manifested as anti-arrhythmia (AF and AVNRT) and anti- ischemia in a rate-dependent manner. Metformin increased excitable index and by increasing A terminated simulated experimental AVNRT. Also, the metformin has a protective effect in AF arrhythmia by an increase in the zone of concealment and increase in the average HH-interval and an increase in the number of concealed beats. Thus, cardioprotective effect of metformin as antiarrhythmia and anti- ischemia in metabolic hepatic diseases mediated via several mechanism is provided.

[0057] Example 2: Effects of Metformin and Non-liposomal Peptide on Preconditioning and Anti-Apoptotic in An Isolated Bile Duct-Ligated Rat Heart

[0058] In Example 2, an administration effect of metformin and non-liposomal peptide on preconditioning and anti-apoptotic in an isolated bile duct-ligated (BDL) rat was investigated, consistent with the teachings of the exemplary embodiments of the present disclosure.

Animals Preparation

[0059] The experiments were performed using adult male Wistar rats (Pasteur Institute of Iran, Iran), with a bodyweight range of 220-260 g. All the animals were kept in animal quarters under standardized conditions with a 12-h light/dark cycle, 20-24 °C ambient temperature, and 45-55% humidity, with free access to rat-food and water according to the guidelines of Golestan University of Medical Sciences.

Bile duct ligation [0060] BDL procedures were performed as described by Liu et aL, (Liu et aL, “Protective effects of erythropoietin on cirrhotic cardiomyopathy in rats. Dig. Liver Dis.”, 44 (12), 1012-1017 (2012), which is hereby incorporated by reference). Briefly, a laparotomy was performed under general anesthesia, induced by a mixture of ketamine and xylazine (100:10 mg/kg). The bile duct was identified and doubly ligated and the cirrhosis model was established after four to five weeks. In sham-operated rats, one loose tie was left in place. Electrocardiogram standard limb lead-ll was monitored before laparotomy procedure (first step) and after 5 weeks, before sacrificing the rats (second step) using a computerized data acquisition system (Lab Chart 7, ADI Instruments, Australia), and analyzed with an ECG analyzer module (Lab Chart PRO v7, ADI Instruments, Australia).

[0061] The biochemical parameters, such as the levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), Bilirubin (Bili), electrophysiological and histopathological data were assessed at the first day of experiments and after inducing cirrhosis. For histopathological parameters, liver samples were collected at the end of experiments from both normal and BDL rats.

Isolated Hearts

[0062] All surgical procedures were performed following an established protocol by Khori et al. (Khori et al., “Acute effects of simvastatin to terminate fast reentrant tachycardia through increasing wavelength of atrioventricular nodal reentrant tachycardia circuit. Fundam. Clin. Pharmacol.”, 29 (1 ), 41-53 (2015), which is hereby incorporated by reference). Briefly, animals anesthetized with a mixture of ketamine and xylazine (100:10 mg/kg) were anticoagulated by heparin (200 lU/kg, IP). The trachea was exposed and connected using a fit cannula to a rodent ventilator (Model 683, Harvard Apparatus). The aorta was cannulated and the hearts were rapidly excised and mounted onto the aortic cannula by a Langendorff Perfusion System at a constant pressure of 80 mmHg with oxygenated (95% 02, 5% CO2, 37 °C) Krebs-Henseleit buffer (pH 7.35-7.45). Metformin and Cyclosporine A were dissolved and equilibrated in Krebs and Henseleit buffer exactly before the experiment and then were perfused with constant pressure via aorta in Langendorff isolated heart model. Bipolar electrograms were recorded (Silver electrodes, 100 pm, AMI Company, Lexington, KY, USA) from the right atrium and the apex. Electrogram signals were filtered (30 Hz-3 kHz) and amplified by ISODAM 8 amplifiers (World Precision Instruments). Data were recorded and analyzed offline by Lab Chart 7 software. Regional ischemia was induced by left anterior descending (LAD) artery occlusion using silk string (6-0 mm), thereby constricting perfused flow from the left ventricular tissue distal to the occlusion (Lochner, Genade and Moolman, 2003a).

[0063] The hearts of all animals were subjected to 30 minutes of regional ischemia and 120 minutes of reperfusion (Fig. 5). Animals were equally divided into eight groups (n=13): (I) l/R: hearts were subjected to 30 minutes of ischemia and 120 minutes of reperfusion, (II) IPC: IPC was induced via four cycles of five minutes of regional ischemia followed by five minutes of reperfusion prior to 30 minutes of main ischemia, (III) l/R (BDL): hearts were subjected to 30 minutes of ischemia and 120 minutes of reperfusion BDL rats, (IV) IPC (BDL): four cycles of five minutes of regional ischemia followed by five minutes of reperfusion prior to 30 minutes of main ischemia in cirrhotic rats, (V) Met (2 pM) was added 40 minutes prior to the main, (VI) Met (BDL): Met was added 40 minutes prior to the main ischemia in BDL rats, (VII) CsA (1 pM) was added 40 minutes prior to the main ischemia, and (VIII) CsA (BDL): CsA was added 40 minutes prior to the main ischemia in BDL rats. EC50 of Met (2 pM) was determined on the l/R injury model in four separate groups in a range of concentrations of 1 *10-6-10x10-6 M. The optimum concentration of CsA was selected based on our previous experiment.

Hemodynamic parameters determination

[0064] All hemodynamic parameters were measured by using a latex water-filled balloon placed into the left ventricle connected to a pressure transducer. These variables were all monitored by Power Lab software (Power Lab 8/30 AD Instruments, Australia). The left ventricular developed pressure (LVDP) (the difference between the left ventricular systolic and diastolic pressures), as a valid and reliable quantitative indicator of contractile function. The rate-pressure product (RPP) was also measured as an indicator of cardiac function (RPP = HRxLVDP - 1000).

Infarct size determination [0065] The infarct size was determined by 2, 3, 5-triphenyl-tetrazolium chloride (TTC) staining as described previously (Alizadeh et al., 2010). The areas of the normal ventricle (blue), the area at risk (AAR, red) and unstained infarcted region (IS, pale) were determined by using the computerized planimetry technique (Photoshop, ver. 7.0, Adobe system, San Jose, CA, USA). The infarct size (IS) and total area at risk (AAR) were expressed as a percentage of the AAR (IS/AAR%) and the left ventricle (AAR/LV%), respectively.

Results

Hemodynamic properties

[0066] Histopathological procedure as well as observing necrosis, formation of fibrotic bridges, and decrease of hepatocytes nuclei depicted in Fig.5 for cirrhotic rats.

[0067] For the functional contractility recovery indexes during reperfusion phase, IPC shows a slight (and not a significant) improvement in LVDP in comparison with l/R group (Fig.6D), whereas IPC (BDL) significantly attenuated LVDP compared to l/R (BDL) (Fig.6D). The results suggested that Metformin significantly succeeded in abolishing this attenuation (P <0.05) (Fig.6C). Similar trends were observed during measurement of RPP (Fig.7), although an improvement of RPP in IPC group was significant this time (P <0.05) (Fig.7D).

Infarct Size

[0068] The effects of BDL were tested on rat hearts undergoing global l/R or IPC with mPTP blocker (CsA) and AMPK activator (Met) on the Langendorff system. The infarct size was decreased significantly from 31 .8 % ± 2.9 for l/R to 12.4 % ± 2.0 in the IPC group (P <0.05) (Fig.8). This significant decrease in the infarct size of the IPC group abolished in IPC (BDL) vs. l/R (BDL) group. Metformin and Cyclosporin’s A significantly decreased the infarct size in the BDL and normal groups (P <0. 05) (Fig.8).

Assessment of arrhythmia

[0069] As illustrated in Fig.9, incidence of VF and arrhythmia score were decreased significantly in the IPC group compared to the l/R group (P <0.05). Addition of cyclosporine’s A decreased incidence of VF in cyclosporine’s A group vs. l/R group (P <0.05). Perfusion of Metformin in the normal and BDL groups significantly decreased arrhythmia score and incidence of VF in comparison to l/R and l/R (BDL) groups (P<0.05) (Fig.9).

[0070] Direct anti-arrhythmia and anti- ischemia effect of metformin and Cyclosporine have a hepatic cardiomyopathy. Metformin anti ischemia effect mediated via increased autophagy and decrease apoptosis by several molecular signaling integrated in the mitochondria. Also, it has protective effect in VF arrhythmia score and incidence by an increase in the zone of concealment and increase in the average RR-interval.

[0071 ] While particular aspects of the present subject matter described herein have been shown and described, it will be apparent to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this subject matter described herein and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this subject matter described herein. Furthermore, it is to be understood that the invention is solely defined by the appended claims. It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should typically be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, typically means at least two recitations, or two or more recitations).

[0072] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first, and second, and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms “comprises,” “comprising,” “include,” “including,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, apparatus, or device that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, apparatus, or device. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or device that comprises the element. Moreover, “may”, “can”, and other permissive terms are used herein for describing optional features of various embodiments. These terms likewise describe selectable or configurable features generally, unless the context dictates otherwise