This study was carried out in accordance with the recommendations of Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NHI, no. 85-23, revised 1996). The protocol was approved by the Institutional Research Ethics Committee of the Nove de Julho University, São Paulo, Brazil (number: 0015–2012). Experiments were performed under ketamine (50 mg/kg), and Xylazine (10 mg/kg) mixture anesthesia, and efforts were made to minimize the suffering of animals.

Figure 1 illustrates the experimental design. Forty-seven female Wistar rats weighing 250–280 g were assigned to LLLT or non-LLLT. The MI was produced by permanent arterial coronary occlusion, and then rats were randomized to one of the following groups: infarcted rats non-treated with LLLT (MI-N, n = 7); infarcted rats submitted to LLLT (MI-LLLT, n = 6). Sham rats (n = 8) were operated upon similarly, although the coronary occlusion was avoided. The Sham and MI-N groups were submitted to a similar LLLT procedure, yet the device was kept off (placebo). Echocardiographic analyses were carried out on 3 days and 5 weeks post-infarction. We have included in the study only rats with large infarcts, which showed be defined on the 3rd-day post-infarction as a size ≥ 37% of LV (dos Santos et al., 2013). At the end of the 5-week, rats were euthanized by decapitation according to a protocol detailed elsewhere (AVMA Panel on Euthanasia. American Veterinary Medical Association, 2001). The infarct scar was removed from LV, and remote myocardial tissue was immediately stored in a cryogenic tube and kept frozen in liquid nitrogen for molecular analysis.

To date, nine rats died in coronary occlusion surgery, four during the peri-operative period, and two in the hemodynamic evaluation. We excluded 11 rats because they had infarct sizes < 37%.

The MI was induced according to a well-established technique (Antonio et al., 2015). Briefly, under anesthesia and artificial ventilation (Harvard Rodent Ventilator, Model 863; Harvard Apparatus, Holliston, MA, USA), a left thoracotomy was performed. The heart was exteriorized, and the left anterior descending coronary artery was occluded near its origin with 6-0 polypropylene. The heart was rapidly returned to its original position and the thorax closed.

Aluminum Indium Gallium Phosphorus—AlGaInP (Twin Laser—MM Optics, São Carlos, SP, Brazil) was used for irradiation under the parameters in Table 1. After thoracotomy, the coronary occlusion was carried out as describe above, and the heart was put in the chest to recover itself for 60 s and then the organs was externalized. The laser/placebo was applied directly to the myocardial tissue targeting infarcted area. In the 3rd day, rats were anesthetized, and a new thoracotomy was performed at the same surgical site to heart exteriorization and laser/placebo application. Sham group was exposed to all experimental procedures, though the LLLT device was off.

Rats were anesthetized as described above (K-X mixture) and LV echocardiography was performed using a 12-MHz transducer connected to an HP Sonos-5500 (Hewlett–Packard, Palo Alto, CA, USA). The infarct size was evaluated on transverse 2-dimensional view and reported as percent of the LV perimeter on the basal, mid transversal, and apical planes (Sofia et al., 2014). The MI was defined as the presence of a segment with increased echogenicity and modification in myocardial thickening or systolic movement (hypokinesia, akinesia, or dyskinesia). Systolic function was analyzed by the fractional shortening (Serra et al., 2010). Diastolic function was not evaluated owing to the fusion of the A and E waves.

Immediately after echocardiography, baseline hemodynamic evaluation was performed under adjusted anesthesia (K-X mixture) and oxygen-enriched ventilation with a closed chest. The left femoral vein was accessed for drug administration, and a 2-F gauge Millar catheter-tip micromanometer (model SPR-320, Millar Instruments, Houston, TX, USA) was inserted into the right carotid artery into the LV cavity. Moreover, an ultrasound flow probe (Transonic System Inc., Ithaca, NY, USA) was positioned in the ascending aorta. The following data were analyzed (Acknowledge software, Biopac System, Santa Barbara, CA, USA): LV systolic (SP) and end-diastolic pressures (EDP), rate of change of LV pressure (+dP/dt and −dP/dt), heart rate, and cardiac output (CO), and stroke volume (SV). Stroke work index (SWI) was stated as previously described (dos Santos et al., 2010). Thereafter, sudden LV afterload increases were achieved using a single phenylephrine in bolus injection (15–25 μg/kg, i.v.) (dos Santos et al., 2010).

After hemodynamic analysis, hearts were quickly removed and weighed. Myocardial mass was indexed by body weight and used as a hypertrophy marker.

Hearts were removed in 3 days and 5 weeks after infarction or sham surgery and fixed in 4% buffered formaldehyde overnight. The LV fragments were washed with PBS, dehydrated through a graded series of ethanol, diaphonized with Xylol and embedded with paraplast. Samples were cut into 3 mm thick sections and stained with Masson's trichome. The fibrous tissue was evaluated in 6 randomized 40 x magnification using a Nikon Eclipse E200 microscope and Nikon Infinity Optical System (Kurobane Nikon Co., Tochigi, Japan), and Image Pro-Plus software, version 4.0 (Media Cybernetics Inc., Rockville, MD, USA).

Proteins were extracted from the LV remote area as previously described by us (Silva et al., 2014). Homogenate protein samples of 30 μg were subjected to SDS-PAGE in 10% polyacrylamide gel. Separated proteins were transferred onto hydrophobic polyvinylidene difluoride membranes (Hybond-P, Amersham Biosciences; Piscataway, J, USA), and the transfer efficiency was examined with 0.5% Ponceau S. The membranes were soaked in a blocking buffer (5% nonfat dry milk and 0.1% Tween 20 in PBS, pH 7.5) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies: rabbit anti-Akt1 (1:5000 dilution; Abcam, Cambridge, MA, USA); rabbit anti-phosphoSer473Akt1 (1:5000 dilution; Abcam, Cambridge, USA); goat anti-VEGF (1:1000; Abcam, Cambridge, MA, USA); anti-GAPDH (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After overnight incubation, membranes were washed five times and then incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit and rabbit anti-goat secondary antibodies (1:2000; Invitrogen, San Diego, CA, USA). Membranes were finally washed five times with blocking buffer and then rinsed twice in PBS. Bound antibody was detected by using chemiluminescence reagent for 1 min. The bands were imaged by using Amersham Imager 600 system (GE Health Care, Little Chalfont, UK). UK).

Data were analyzed using GraphPad Prism software 5.0 (La Jolla, CA, USA). Shapiro-Wilk test was used to verify normality data. Levene test was applied to assess the equality of variances. One-way ANOVA complemented by Newman–Keuls post hoc was applied to detect differences between groups in cross-section analysis. Two-way repeated ANOVA complemented by Bonferroni post hoc was applied to paired data. Kruskal-Wallis followed by Dunn's multiple comparison tests were applied to non-normality data. Statistical significance was set at p ≤ 0.05. Data are expressed as mean ± SD.

The biometric data are shown in Table 2. Average body weight was similar between the three experimental groups on the 3rd day and 5 weeks of study. Infarcted rats showed a similar heart mass as well as heart mass-to-body weight ratio when compared with Sham rats, and phototherapy had no repercussion on heart mass. Besides, phototherapy also had no effect on infarct size. As evidenced in Figure 2, quantitative analysis for Masson trichome staining indicated no significant differences in the collagen content between experimental groups with 3 days post-MI. However, infarcted rats showed a significant increase of fibrosis over 5 weeks post-MI, in which LLLT had no significant effect.

As seen in Figure 3, there was LV dilatation with only 3 days post-infarction, in which diastolic diameter was significantly higher in MI+LLLT group while the systolic diameter was higher in all infarcted groups when compared with Sham group. At the end of the 5-week experimental period, both diastolic and systolic diameters were shown to be significantly increased in all infarcted groups when compared with Sham group. The LV systolic dysfunction was apparent early as the 3rd-day post-infarction, as evidenced by a minor fractional shortening. The beneficial role of LLLT was only noticed in the early (3 days) post-infarction cardiac remodeling, in which the fractional shortening of the MI-LLLT group was significantly higher than MI-N group. On the other hand, treated-LLLT rats show a lower LV performance in the 5th week of study when compared with Sham and MI-N rats.

Afterward second echocardiographic analysis, an invasive hemodynamic evaluation was carried out to determine LV ejection performance. As reported in Figure 4, data also indicate deteriorating LV function, in which +dP/dt and −dP/dt values were significantly lower in MI-N and MI-LLLT groups compared with Sham group under basal conditions. In addition, a higher EDP was reported only for MI-LLLT group, which also showed a more marked reduction on −dP/dt when compared to MI-N and Sham group. The LV ejection parameters from all infarcted groups did not differ significantly from those of the Sham group when evaluated under basal conditions, as evidenced by SWI and CO. These findings led to analyze the cardiac functional reserve during sudden afterload stress as a result of in bolus phenylephrine injection. For suitable homogenization, we carried out experiments to raise the blood pressure of 50–70% over the baseline level (dos Santos et al., 2010). This afterload range was accompanied by a higher increase of +dP/dt and −dP/dt in Sham group than in all infarcted groups. Furthermore, CO decreased more dramatically in all infarcted groups when compared with Sham group. Ultimately, Sham rats showed SW increase, whereas the SW was remarkably reduced in all infarcted rats.

It has been postulated that the cardioprotective effects of LLLT shown to be associated with increased angiogenesis, and this action is linked to modulation of vascular endothelial growth factor (VEGF) (Tuby et al., 2006, 2008). Thus, we have investigated the Akt₁/VEGF pathway in the remote myocardial after 5 weeks following injury. Data in Figure 5 indicate that the MI and phototherapy did not affect the expression of the total Akt₁, Akt₁ phosphorylated at Serine 473 and Akt₁/pAkt₁ ratio, which is a marker of its activity. Notwithstanding, VEGF expression was also not significantly different between the experimental groups.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.