Interaction of exercise training with taurine attenuates infarct size and cardiac dysfunction via Akt–Foxo3a–Caspase-8 signaling pathway

This research aimed to investigate the synergistic protective effect of exercise training and taurine on Akt–Foxo3a–Caspase-8 signaling related to infarct size and cardiac dysfunction. Therefore, 25 male Wistar rats with MI were divided into five groups: sham (Sh), control-MI(C-MI), exercise training-MI(Exe-MI), taurine supplementation-MI(Supp-MI), and exercise training + taurine-MI(Exe + Supp-MI). The taurine groups were given a 200 mg/kg/day dose of taurine by drinking water. Exercise training was conducted for 8 weeks (5 days/week), each session alternated 2 min with 25–30% VO2peak and 4 min with 55–60% VO2peak for 10 alternations. Then, the left ventricle tissue samples were taken from all groups. Exercise training and taurine activated Akt and decreased Foxo3a. Expression of the caspase-8 gene was increased in cardiac necrosis after MI, While, after 12 weeks of intervention decreased. Results exhibited that exercise training combined with taurine has a greater effect than either alone on activating the Akt–Foxo3a–caspase signaling pathway (P < 0.001). MI-induced myocardial injury leads to increase collagen deposition (P < 0.001) and infarct size and results in cardiac dysfunction via reduced stroke volume, ejection fraction, and fractional shortening (P < 0.001). Exercise training and taurine improved cardiac functional parameters (SV, EF, FS) and infarct size (P < 0.001) after 8 weeks of intervention in rats with MI. Also, the interaction of exercise training and taurine has a greater effect than alone on these variables. Interaction of exercise training with taurine supplementation induces a general amelioration of the cardiac histopathological profiles and improves cardiac remodeling via activating Akt–Foxo3a–Caspase-8 signaling with protective effects against MI.


Introduction
Cardiac diseases are the major reason for morbidity and mortality worldwide which is a well-accepted fact . Myocardial infarction (MI) is a term used for a heart attack, a kind of cardiac disease that results in heart muscle injury (Lu et al. 2015), pathological changes in the left ventricular (Maessen et al. 2017), and cardiac structure and function deterioration (Wang et al. 2020). MI leads to increased cell death, an increase in the infarct area and scar formation, and a decrease in cardiac function (Sengupta et al. 2011). Studies demonstrated that the activation of FoxO3a has a pivotal role in cardiomyocyte apoptosis after MI and, leads to increased proteasomal proteolysis, thus contributing to contractile proteins degradation (Zhang et al. 2018).
Akt has activated via PI3K phosphorylation, activated Akt leads to phosphorylation and nuclear export of Foxo3a, and as a result, the functional inactivation of Foxo3a (Tzivion et al. 2011), because the accumulation of Foxo3a in nuclei will induce the Caspase-8. Foxo3a regulates the expression of caspase-8 (Geiger et al. 2012). As findings show, the functional inactivation endogenous of Foxo3a leads to reduce endogenous expression of caspase-8. Phosphorylated Foxo3a can attenuate myocardial necrosis by suppressing caspase-8 (Maiese et al. 2009).
There is evidence that apoptosis and necrosis have common pathways, and in the pathology of the myocardium, it is difficult to distinguish between these two cell death pathways. In fact, in addition to their well-established role in apoptosis, caspases may also mediate necrotic injury. Thus, caspase-8 inhibition can prevent myocardial cell death after MI (Mocanu et al. 2000;Denecker et al. 2001;Wang et al. 2003;Vandenabeele et al. 2006;Yuan et al. 2016).
Exercise training is an effective intervention in the prevention and treatment of cardiac disease. Exercise training in cardiac diseases, particularly, in patients with MI improves the quality of life and reduces hospitalization and fatality. Animal studies have shown that regular exercise training after myocardial infarction increases cardiac remodeling and maintains cardiac function (Lee et al. 2018). Wang et al. (2020) found that high-intensity interval training after MI for 8 weeks preserved cardiac functions, decreased infarct size, protected the heart structure, reduced collagen accumulation, and contributed to myocardial repair (Wang et al. 2020).
Moreover, recent findings have established amino acids as contributing factors in failing hearts with a potential impact on pathological remodeling and dysfunction in the heart (Sun and Wang 2016). One of the important amino acids whose deficiency leads to severe pathology and cell death is taurine which is the most abundant functional free amino acid in a very high concentration in the cardiac tissue (Jong et al. 2017). Decreased taurine concentration is associated with the occurrence of impairment and cardiac pathologies (Qaradakhi et al. 2020). Taurine prevents induced-MI cardiomyocyte apoptosis (Li et al. 2009;Zhang et al. 2013), but deficiency promotes cardiac apoptosis (Jong et al. 2017). Taurine content diminished when cardiac tissue was involved in hypoxic or ischemic stress, while administration of taurine elevated the taurine levels in ischemic cardiac tissues (Zhang et al. 2013). Thus, taurine is an important nutrient that improved left ventricular function parameters and reduces structural abnormalities in heart muscle (Mele et al. 2019). Taurine also is an ergogenic supplement in exercise training that serves as an anti-inflammatory agent, improved aerobic, and aerobic performance and recovery, and reduced metabolic markers (Kurtz et al. 2021). Ueno et al. (2007) have reported administration of taurine decreases myocardial injury and preserves functional parameters in rat hearts (Ueno et al. 2007). Liu et al. (2020) demonstrated taurine mitigates cardiac dysfunction by decreasing myocyte hypertrophy and fibrosis and reducing apoptosis and oxidative stress .
However, the combination of taurine and exercise could be effective in cardiac functional parameters after myocardial infarction but the basic mechanisms have not been determined. Our research highlights a new model in which combined exercise training and taurine involve a signaling pathway related to Akt, Foxo3a, and caspase-8. The extent of their expressions may provide a new non-pharmaceutical way to manage myocardial necrosis.

Ethical approval
All experiment procedures were approved via the Ethics Committee of Sport Sciences Research Institute (IR.SSRI. REC.1400.1303) and conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th ed., 2011) (Lee et al. 2018).

Animal model
Male Wistar rats weighing 300-350 g, 12 weeks old, were purchased from Baqiyatallah University of Medical Sciences and were housed at a temperature (23-25 ℃) and humidity (55%) on a 12 h dark/light cycle (dark on 08:00-20:00 on each day), with free access to chow diet and water all over the experiment. After a week of feeding, rats were subjected to MI and Sham surgery, an echocardiography was performed.

MI surgery
The MI model was created by ligating the LAD coronary artery. Animals were anesthetized with Ketamine (150 mg/ kg,ip) and xylazine (15 mg/kg,ip). From the fourth intercostal space surgical incision into the chest wall was performed and the LAD was ligated with a 6-0 silk suture. The Sham group underwent the same method except for ligation of the LAD, and no infarction was created. It should be noted that surgery was performed by a veterinarian at the Experimental Research Center of Cardiovascular Hospital.

Assessment of cardiac function
Echocardiography measurements were performed 1 and 12 weeks after MI surgery. Rats were anesthetized for echocardiography according to the conditions mentioned in the MI surgery ward. In this process, the left ventricle was used to calculate the stroke volume (SV), LV injection fraction (%EF), and LV fractional shortening (%FS). The rats with a high grade of myocardial damage, estimated at FS ≤ 35%, were subjected to the study (Kraljevic et al. 2013).

Exercise training
Survival rats post-MI surgical and EKG were divided into five groups: sham group (Sh; n = 5), control-MI group (C-MI; n = 5), exercise training-MI group (Exe-MI; n = 5), taurine supplementation-MI group (Supp-MI; n = 5), and exercise training + taurine-MI group (Exe + Supp-MI; n = 5). Exercise training moderate intensity interval training (MIIT) using a motor-driven treadmill at a 0° incline was conducted for 8 weeks (5 days/week). For familiarization of rats with the training stress conditions, 2 weeks after MI surgery, exercise training was involved in an adaptive period at 10 m/ min for 10 min per day at a 0° incline, for 5 successive days and 2 free days. Then the Wisloff protocol was used to determine VO2peak (Høydal et al. 2007). The first formal exercise training session in Exe-MI and Exe + Supp-MI groups was initiated 4 weeks after recovery from the MI surgery. Rats alternated between 4 min with 55-60% VO2peak and 2 min with 25-30% VO2peak for 10 alternations, for 5 consecutive days and 2 free days (Kraljevic et al. 2013). The whole time exercise training was 60 min per day. The Sham, C-MI, and Supp-MI groups remained sedentary all over the research period ( Fig. 1).

Oral taurine administration
Taurine in powder form with purity > 99% was purchased from Sigma Aldrich. Each rat was housed in a separate cage. Rats were treated from the fourth week to the twelfth-week post-MI (8 weeks) with oral taurine via drinking water. It was administered at a dose of 200 mg/kg body weight per day, according to the rat's mean body weight and daily water consumption (Jia et al. 2016). For the whole duration of the study, water consumption was monitored and recorded; with this method, it was ensured that the rats consumed oral taurine (Fig. 2).

Masson's trichrome staining
After sacrificing rats, the removed heart tissue was washed in ice-cold saline and was kept at − 80 °C up to further analysis. Then, the right and left ventricles were separated. To measure infarct size, the samples were fixed in ice-cold 4% Paraformaldehyde for 48 h, and afterward embedded into Paraffin blocks and cut into sections of 5 μm thickness. This technique was used to assess myocardial collagen deposition, and collagen volume fraction (CVF) with Image-Pro plus analysis software was measured. The ratio of infarct was quantified as CVF (Song et al. 2020).

Western blot analysis
This method was done to measure the expression levels of Foxo3a and Akt proteins. The samples of the non-infarcted left ventricle area (near the infarcted zone) were prepared and then initial samples were homogenized. First, the collected samples were centrifuged at 10,000 g for a period of 30 min, and supernatants were obtained. The diluted samples were treated at 95 ℃ heat for 5 min and separated by sodium dodecyl sulfate-polyacrylamide gel and transferred to Poly-vinylidene fluoride membranes. Membranes were pre-incubated in tris-buffered saline for 1 h and then incubated with specific primary antibodies for Akt (#9272), Foxo3a (SC-48348), and GAPDH (GTX100118) overnight. After washing antibodies in TBS with Tween 20, the membranes incubation was done by using horseradish peroxidase-conjugated secondary antibodies. [Secondary (rabbit): BA1054-2, Secondary (mouse): SC-516102] Following this, the enhanced chemiluminescence (ECL) method was used to see proteins. Finally obtained films were analyzed by image-J software (NIH, USA) ).

Fig. 1
Schematic description of experimental design and protocol of exercise training after MI surgery in rats. 2 weeks after MI or sham surgery, the rats were subjected to adaptive exercise training for 2 weeks and to formal exercise training and taurine supplementation for extra 8 weeks. All animals were euthanized for heart tissue sample collection at the end of the protocol, that is, 12 weeks after MI or sham surgery

Real-time quantitative PCR
To characterize whether exercise training and taurine after MI affect Caspase-8, mRNA expression related to Caspase-8 was assessed in the left ventricle. Non-infarct zones of the left ventricle were homogenized with one ml QIAzol Lysis Reagent (Qiagen, NO; 79306, USA), and total RNA was extracted. cDNA was synthesized from 10 µg DNase I-treated total RNA by reverse transcription with Revert Aid First Strand cDNA Synthesis primers (Thermo Scientific, NO; K1622, USA) and Superscript II RNase H reverse transcriptase (Thermo Scientific, NO; K1622, USA) (Table 1). RT-PCR was administrated using Real Q plus Master Mix Green high ROX™(No. 4309155, Amplicon, Denmark) with SYBER Green I Method. The PCR reaction was normalized to the GAPDH reference gene. The following formula was also used to determine gene expression.

Statistical analyses
The values were shown as mean ± SEM and data were analyzed with SPSS 21.0 software. To compare groups, one-way ANOVA was used, and then Tukey's post hoc test was performed. Mix method (Repeated ANOVA between subjects) was done to evaluate changes in SV, EF, and FS. P ≤ 0.05 was considered significant.

Bodyweight
Rats were monitored for body weight. The body weight of rats was important throughout the research because calculating the dose of taurine was based on its drinking water. The results for bodyweight measurements at 12 weeks are reported in Table 2.

Histopathology of cardiac tissue
The effects of exercise training and taurine supplementation on the cardiac structure are shown in Fig. 3a, b. As Masson's trichrome staining revealed, there was excess fibrosis and disarranged cardiac muscle fibers in the C-MI group (blue staining), and histopathological signs including collagen deposition and cardiomyocyte degeneration were observed in the C-MI group heart. In special, large areas of necrotic myocytes were observed. While, there was a decrease in these histopathology signs, in groups of Exe-MI, Supp-MI and Exe + Supp-MI. Statistical analysis revealed that combined taurine and exercise training improved histopathological lesions in cardiac muscle. In specific, a significant decrease in collagen deposition was observed in intervention groups with respect to the control group (Fig. 3).

The effect of exercise training combined with taurine on the Akt-Foxo3a pathway activation
The Akt-Foxo3a pathway appears to promote the growth of the myocytes and inhibits infarction myocardial-induced necrosis. We tested whether exercise training and taurine were capable of activating the Akt-Foxo3a signaling pathway. To accomplish this, the expression of Akt protein in the heart tissue was measured by Western blot. We found a remarkable decrease of Akt protein levels in the C-MI group cardiac tissue after infarction myocardial, compared to sham (Fig. 4A), while its expression levels increased in other groups compared to the C-MI group. This suggests that exercise training and taurine could activate Akt protein; Also, the relative levels of Akt protein in the Exe + Supp-MI group had a significant increase compared to other study groups including Exe-MI and Supp-MI groups, which exhibits that exercise training combined with taurine has a greater effect than either alone on activating the Akt-Foxo3a signaling pathway.

Increased expression of Foxo3a in necrotic cardiac tissue after MI
To explore the role of Foxo3a, protein expression of Foxo3a in the cardiac tissue was evaluated by Western blot. As displayed in Fig. 4B, protein expression of Foxo3a presented a significant increase in the C-MI group, compared to sham. While expression of this protein was decreased in the Exe + Supp-MI, Exe-MI, supp-MI groups after 12 weeks of intervention (4 weeks recovery, 8 weeks exercise training + taurine supplementation); and, this effect was greater in the Exe + Supp-MI group than either alone.

The effect of exercise training combined with taurine on the expression of Caspase-8 in myocardial necrosis after MI
A cysteine-protease that has an important function in regulating cellular apoptosis is Caspase-8. It has recently been demonstrated that it is critically involved in regulating cardiac necrosis. Expression of caspase-8 in the cardiac tissue was measured by RT-qPCR. We observed that gene expression of caspase-8 was increased in cardiac necrosis after MI. As shown in Fig. 4D, gene expression of caspase-8 was remarkably increased in the C-MI group, compared to the sham group. While, after 12 weeks of intervention (4 weeks of recovery, 8 weeks of exercise training + taurine supplementation), its expression has decreased in the Exe + Supp-MI, Exe-MI, Supp-MI groups compared to C-MI group. Besides, the gene expression levels of caspase-8 in Exe + Supp-MI group had a significant decrease compared to Exe-MI and Supp-MI groups. It suggests that more suppression of the expression of caspase-8 took place by training and taurine than either alone. Collectively, exercise training and taurine supplementation reduced caspase-8 activity.

Exercise training combined with taurine improves post-MI cardiac function
After 12 weeks of intervention, the anesthetized rats, before sacrificing, underwent hemodynamic measurement of cardiac functional parameters. Results are shown in Fig. 5. Selected cardiac functional parameters were impaired in the C-MI group, as evidenced by a remarkable decrease in stroke volume (SV), LV ejection fraction (%EF), and LV fractional shortening (%FS) when compared with the sham group. In contrast, compared with the C-MI group, exercise training and taurine resulted in a remarkable increase in SV, %EF, and %FS after 12 weeks of intervention. Collectively, functional parameters were enhanced via 8-week exercise training combined with taurine, and remarkable differences among groups were observed for the major parameters relevant to left ventricular function (Fig. 5A, B, C, D, E, F).  (Chen et al. 2021). As echocardiography data indicated, the increased infarct size resulted in cardiac dysfunction via reduced stroke volume, ejection fraction, and fractional shortening. On the other hand, MI-induced myocardial injury was improved via modifying Akt, Foxo3a, and Caspase-8, and collagen deposition was reduced. Our data demonstrated that MI-induced myocardial injury can be attenuated via 8 weeks of exercise training combined with taurine supplementation. We found more improvement in cardiac functional parameters under the administration of taurine and exercise training concomitantly. Activation of Akt is a cytoprotective pathway in that a host of agents, including nutrients and exercise training, take their anti-apoptotic function via the activation of Akt (Schaffer et al. 2014;Jia et al. 2019).
Taurine supplementation (Sun et al. 2013;Sedaghat et al. 2020) and exercise training (Cai et al. 2018;Lee et al. 2018;Jia et al. 2019) induce up-regulation of activated Akt, which we observed in this study. Exercise training after MI via increasing SIRT1 and PGC-1α expression leads to an activated Akt-Foxo3a signaling pathway (Jia et al. 2019). Akt prevents cellular apoptosis via the phosphorylation of the Foxo3a protein (Lin et al. 2014). Activated Akt translocates from cytoplasm to nucleus and phosphorylates Foxo3a. Then, Foxo3a phosphorylation results in the translocation of Foxo3a from the nucleus to the cytoplasm and causes a reduction in its transcriptional activity via negative feedback (Fasano et al. 2019). The outcome of Foxo3a phosphorylation by Akt attenuates Foxo3a transcriptional activity and increases degradation (Tzivion et al. 2011). Whereas, Foxo3a accumulation in the nucleus will induce the Caspase-8 and cause cell apoptosis (Lin et al. 2014). Caspase-8 is the originator caspase of extrinsic apoptosis. The results of previous studies revealed the role of enzymatic activity and function of caspase-8, which leads to the activation of the inflammasomes (cytosolic complexes that process the pro-inflammatory cytokines into their biologically active forms) and induction of pyroptosis (Fritsch et al. 2019). Orning P. and Lien E. (2021) revealed that caspase-8 plays an important role in cell death and inflammation (Orning and Lien 2021). Caspase pathways are related to the transcription of FoxO3a because the increased activity of FoxO3a results in caspase-induced apoptotic death (Maiese et al. 2009). The results demonstrated that the expression of the caspase-8 protein was much higher in the control group than that in other groups. Our data thus suggest that exercise training combined with taurine supplementation inhibited caspase-8 activation in the heart of rats.
The results of some research suggest that exercise training and taurine can diminish oxidative stress and apoptosis via activating the Akt and improve the cardiac structure and function after MI Bo et al. 2021).
Taurine also is a strong antioxidant. It can improve exercise capacity (Ahmadian et al. 2017), the reason for this can be explained by Starling's effect due to improved cardiac Exe+Supp-MI Fig. 3 (continued) contraction, which results in better cardiac output (Suwanich et al. 2013).
Exercise training (Lin et al. 2014;Cui et al. 2020) and taurine (Taranukhin et al. 2008;Sun et al. 2013) lead to the activation of Akt and inactivation of Foxo3a and suppression of caspase-8 apoptotic signaling. The exercise training combined with taurine supplementation confirmed both interventions inhibit cardiac apoptosis following MI by the Akt-Foxo3a-Caspase-8 signaling pathway.
On the other hand, after myocardial infarction, a relative lack of oxygen and nutrients may be an important factor in myocardium death (Zhang et al. 2009), which may contribute to increased scarring and fibrosis, and result in loss of cardiac function. Furthermore, the fractional shortening, ejection fraction, and cardiac parameters are reduced after MI, whereas collagen volume is increased (Luther et al. 2012), and the elastic fiber is decreased gradually (Yu et al. 2018). Kinetics of decreased elastic fiber (Cowling et al. 2019) in combination with increased collagen results in great resistance to distension (Zhang et al. 2009), increased stiffness, and decreased flexibility of the myocardium, which normal cardiac function will be reduced, and may process to cardiac dysfunction and even heart failure (Yu et al. 2018). The cell types that have been involved as responsible for collagen secretion in the diseased heart include myofibroblasts, cells derived from EMT, inflammatory cells, and infiltrating fibrocytes (CD34 + , CD45 +) (Cowling et al. 2019).
On the other hand, Masson's trichrome staining showed severe collagen accumulation in the control group, which indicated necrosis (Infarct size) in the heart tissue that resulted in a decrease in cardiac functional parameters (EF, FS, SV). Also, Collagen accumulation in the group of exercise training combined with taurine supplementation was significantly reduced in the necrotic areas. MIinduced Foxo3a activation reduces the size and weight of the heart and affects the structure of cardiomyocytes resulting decrease stroke volume, due to the reduction of the ejection fraction (Schips et al. 2011).
There is a linear relationship between infarct size and levels of intracellular taurine and its restoration of the intracellular taurine pool could prevent an increase in infarct size (Ueno et al. 2007;Schaffer et al. 2014). Therefore, it is rational to suppose taurine supplementation sustains normal levels of taurine in the mitochondria and minimizes mitochondrial ROS production, which is the main cause of myocardial infarction injury (Ueno et al. 2007;Li et al. 2009;Fig. 4 Effect of MI, exercise training, and taurine on Akt (A) and Foxo3a (B) relative protein levels and Western blots of Akt and Foxo3a proteins (C) and gene expression of Caspase-8 (D)in the peri-infarcted zones of the heart.(Sham, C-MI group; Exe + supp-MI group; Exe-MI group, and Supp-MI group. The results are mean ± SD (n = 5/group), *P < .05; **P < .01; ***P < .001) ▸ Schaffer et al. 2014). Mele et al. (2019) found that taurine improved cardiac dysfunction, with a significant decrease area of damage (Mele et al. 2019). Jing Liu et al. (2020) found that taurine therapy increases intracellular taurine concentrations in the heart, improves markers of cardiac function (%EF, %FS), prevented deterioration in cardiac function, and also decreases the expression of apoptosisrelated proteins (caspases). In addition, taurine therapy markedly reduced cardiac fibrosis ). Our knowledge is based on limited data regarding the evaluation of taurine in MI.
High-intensity interval training (HIIT) decreases cardiac apoptosis markers, increases the anti-apoptotic protein expression, and reduces infarction size (Shahrabadi et al. 2022). Cardiac output and stroke volume ameliorate in response to exercise training (Brown et al. 2003). Mechanisms of this effect include decreased catecholamine secretion, mitochondrial dysfunction, ROS production, and increased activated Akt (Fatahi et al. 2022;Ranjbar 2022). Zhang et al. (2021) demonstrated that HIIT and aerobic training increased %EF and improved exercise capacity in rats with MI (Zhang et al. 2021). Lu et al. (2015) indicated that %EF, %FS, exercise capacity, and cardiac function were improved following HIIT and aerobic exercise training and two forms of exercise training decreased apoptosis of the post-MI (Lu et al. 2015). This suggests that intervention factors enhance cardiac function presumably via the Akt-Foxo3a-Caspase-8 signaling cascade pathways in cardiac tissue.
As far as our knowledge, this is the first study that perused the effect of exercise training combined with taurine on infarct size and cardiac dysfunction via the Akt-Foxo3a-Caspase-8 signaling pathway. Our results indicate that the synergistic effect of exercise training and taurine supplementation is effective in improving clinical indicators in patients with MI and can reduce the infarction size and is effective in improving the conditions of heart patients and their quality of life.
Likely because taurine and exercise training are key determinants of contractile function, those interventions increased phosphorylation of Akt and Foxo3a, reduced caspase-8, and finally improved cardiac function. Thereby preventing impairment of cardiac function, apoptosis, and necrosis of cardiac tissue (Lin et al. 2014;Schaffer et al. 2014). These findings could suggest a possible positive role for exercise training and taurine in the activation Akt signaling pathway.
The main limitation of the present research is the fact that we did not measure taurine concentration changes and the extent of taurine depletion in cardiac tissue. Further data collection including taurine concentrations in the heart would be needed to determine the exact beneficial effects of cardiac MI.
In summary, our findings reveal that exercise training combined with taurine has protective effects against MI via improving cardiac function. The mechanisms behind this may include activation of Akt and inactivation of Foxo3a, reduction of inflammation, decrease in collagen deposition, and infarct size. These results provide a new strategy to improve MI-related pathological changes.

Conclusion
Interaction of exercise training and taurine, post-MI, can improve cardiac contractions, reduce fibrosis, necrosis, and cardiac dysfunction, and, it can improve left ventricular function. Taken together, this study suggested that exercise training combined with taurine improves pathological remodeling and cardiac functional parameters via activating the Akt-Foxo3a-Caspase-8 signaling pathway. We propose that the administration of exercise training and taurine supplementation in cardiac patients outlines a novel mechanism that could be an excellent opportunity to improve both their life quality and expectancy.