When to Use Cold Laser Treatment Myocardial Infarction？

Author: May

Sep. 09, 2024

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Effect of low-level laser physiotherapy on left ventricular ...

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Low-level laser therapy may add benefits to improve symptoms, clinical condition, and quality of life in patients with left ventricular systolic dysfunction, further studies are necessary to evaluate the changes in cardiac functions at a longer follow-up duration.

Of the total patients enrolled in the study, 21 (or 77.8%) were male, with a mean age of 57.7 ± 6.89 years. NYHA classification significantly improved after low-level laser therapy, 15 patients were in class III,12 were in class IV, and no one was in class II before laser therapy while after laser therapy; 25 patients shifted to class II, two patients were in class III with P < 0.001, Six-minute walk distance test was performed, and the results showed that the mean of 6MWT was less than 200 m (148.556 ± 39.092) before the study but increased to more than 300 after laser therapy (385.074 ± 61.740), left ventricular ejection fraction before laser therapy was 26 ± 7.5 while after laser therapy it became 30 ± 8.6 but diastolic function did not change after low-level laser therapy, the mean peak TR pressure was 40.0 ± 9.0 mmHg and 33.0 ± 7.0 before and after laser therapy respectively P < 0.001. A significant change was observed in NO level from 4.1 ± 1.4 IU/ml before laser therapy to 5.2 ± 1.7 IU/ml after laser therapy P < 0.001.

Low-level laser therapy (LLLT) is a promising noninvasive physiotherapeutic approach that has been demonstrated to improve cardiac performance. This study aimed to assess the impact of low-level laser therapy on cardiac functions and clinical status in patients with chronic left ventricular systolic heart failure who were not candidates for cardiac revascularization or resynchronization. A case series of 27 patients received a course of low-level laser physiotherapy, the clinical outcomes, echocardiographic parameters, and serum nitric oxide levels were evaluated before and after LLLT.

Based on the aforementioned rationale, we hypothesized that cardiac functions might be improved after LLLT; therefore, we sought to compare the echocardiographic parameters, clinical characteristics, and serum nitric oxide levels in patients with chronic systolic heart failure before and after LLLT.

Furchgott's pioneering work was later expanded and validated by other investigators, who also demonstrated how light might affect the localized generation or release of NO and trigger vasodilation by way of the impact of NO on cyclic guanine monophosphate (cGMP) [ 14 ]. According to these studies, lighting devices with suitable designs could serve as efficient, noninvasive therapeutic agents for individuals who would benefit from elevated NO levels [ 12 , 15 ].

Nitric oxide (NO) may be photo-released from extra intracellular storage, such as nitrosylated hemoglobin and nitrosylated myoglobin, in addition to being photo-dissociated from Cox [ 11 ]. Furchgott first identified light-mediated vasodilation in while working on the nitric oxide project that would earn him the Nobel Prize [ 12 , 13 ].

Laser therapy has a cardioprotective effect, which makes it beneficial for patients with exertional angina and has positive effects on patients' overall health, as seen in a reduction in the incidence of anginal attacks and episodes of myocardial ischemia without pain, and this is accompanied by other beneficial changes in hemodynamic status [ 8 , 9 ]. A substantial decrease in pain-free episodes of myocardial ischemia, which is a prognostically beneficial fact, is one indication that laser therapy had an impact on the relationship between painful and painless ischemia of the myocardium [ 10 ].

Laser therapy has been proven to substantially reduce systolic, diastolic, and mean arterial pressure [ 4 ]. Moreover, submaximal cycling exercise was also found to have a favorable hypotensive effect, with no increase in diastolic arterial pressure and a decrease in total peripheral vascular resistance [ 5 , 6 ]. The use of laser puncture allowed patients to use less hypotensive medications and allowed physicians to prescribe laser therapy regardless of the patient&#;s hemodynamic parameters [ 7 ].

Low-Level Laser Therapy (LLLT) has emerged as a promising physiotherapeutic technique since it is noninvasive and relatively inexpensive. It has been shown that LLLT enhances cardiac function and myocardial contractility, decreases blood pressure, and improves myocardial, coronary, and aerobic reserves [ 1 , 2 ]. This clinicofunctional efficacy was accompanied by modifications that were favorable to lipid metabolism, lipid peroxidation activity, antioxidant defence, hemocoagulation, and microcirculation [ 3 ].

Categorical data were presented as numbers and proportions. Paired categorical data was compared using the Mcnemar test. Normality testing was conducted using the Shapiro-walk test. Continuous variables that were normally distributed were presented in terms of mean and standard deviation and compared using paired t-test. The median and interquartile range were shown for non-normally distributed variables. All statistical tests with a value of P < 0.05 were considered statistically significant. All statistical analyses were done with Statistical Package for Social Sciences (SPSS), version 26 (SPSS Inc, Chicago, IL).

Patients were subjected to low level laser therapy using a therapeutic unit (Phyaction CL) with wavelength 905 nm, output 5&#;20 MW, laser beam spot size 0.785 cm2, energy density 91 J/cm 2 , and energy delivered 28 J, and frequency 500HZ (Fig. ). The laser wavelength was chosen accordingly significant effects of LLLT were previously reported [ 17 ]. Laser probe was placed on the intercostal space corresponding to the myocardium lesion both anteriorly and posteriorly on the chest wall and arm with standardized laser acupuncture points of application. The patient laid in the supine position, with hip and knee joints flexed and feet rested on the bed in a relaxed position. The site of application of laser therapy must be on clean skin. Each acupuncture point was stimulated with a laser for 60 s, once daily, with the frequency of five weekly sessions for two successive weeks. Acupuncture points are anterior chest wall LU1, LU2, CV17, (Ren17), posterior chest wall from a prone or setting position of the patient UB13, (BL13), UB 17(BL17), and arm UL5, UL (Fig. A, ).

Using the transthoracic cardiac probe (X5-1) with tissue Doppler capability from the Philips iE33 machine, echocardiographic evaluation was performed both before and after the investigation. To evaluate the systolic and diastolic function of the left side of the heart, 2-D echocardiography, and tissue doppler imaging (TDI) are used to assess the left ventricular function. Systolic function was evaluated using geometry (modified Simpson method), the operator tracing the end-diastolic and end-systolic volumes, and the machine then automatically calculated the ejection fraction on the software using the formula: ejection fraction = (EDV &#; ESV)/EDV. Septal E/e' was used to assess diastolic function in addition to assessing E wave mitral deceleration time. Mitral regurge was evaluated by the regurgitant jet area/left atrial region, vena contracta, and mitral regurgitant jet area. Vena contracta greater than 5.0 mm regarded severe, 3&#;5 mm moderate, and less than 3.0 mm considered mild mitral regurgitation [ 16 ], whereas more than 40% considered severe mitral regurgitation, 20&#;40% considered moderate, and less than 20% considered mild mitral regurgitation. Applying a continuous wave doppler cursor in alignment with tricuspid regurgitation allowed for the measurement of the peak tricuspid regurgitation pressure, which was 4V 2 . All echocardiography parameters were performed by one cardiologist.

Patients who were older than 18 years old were diagnosed with systolic heart failure based on clinical presentation and confirmed on echocardiography. Patients with a left ventricular ejection fraction (LVEF) < 45% and New York Heart Association (NYHA) classes III and IV, Patients who were not candidates for CRT or revascularization as well as those in NYHA Classes IV or III or II who were receiving optimal medical care and had been hospitalized for HF during the previous 12 months were included in the study. Patients who had been scheduled for (CABG) or percutaneous coronary intervention (PCI) during the previous 90 days or CRT were excluded. Other exclusion criteria included pregnancy, malignancy, thyroid illness, epilepsy, LBBB with QRS 130 ms, and contraindications to laser therapy. All patients were subjected to optimization of the medical treatment for three months before the study. Each patient received 10 sessions of low-level laser therapy. The measurement parameters were directly before the first session and following the last session. Blood samples were withdrawn before and after the laser therapy for measuring serum nitric oxide by the enzyme-linked immunosorbent assay (ELISA). NYHA classification was assessed for all patients before and after the study. All patients underwent the six-minute walk test (6MWT) both before and after the research, with standardized instructions and encouragement. Patients were told to move as quickly as they could while walking as far as they could. They were informed that, if required, they might slow down or even halt. As with the hallway test, the patient was allowed to rest or stop at any time before or during the treadmill walk test. The walk test was also stopped if the patient experienced chest pain, unbearable dyspnea, cramps, disorientation, diaphoresis, or pallor.

A case series of 27 consecutive adult patients with chronic systolic heart failure secondary to ischemic heart disease was conducted at Critical Care Department, Cairo University Hospitals between May , and December . The Declaration of Helsinki's guidelines were followed during the study. The Ethics Committee and institutional review boards of both the Critical Care Medicine Department and Faculty of Pharmacy, October 6 University examined and approved the study protocol and informed consent form. All patients who participated in the trial provided written fully informed consent.

The mean regurge area/left atrial area was 27.0 ± 9.0 before laser therapy and 26.0 ± 9.0 after laser therapy with non-significant P = 0.357 denoting moderate mitral regurgitation that wasn&#;t affected by laser therapy. Another parameter was assessed to detect the severity of mitral regurgitation; the regurge vena contracta (V.C), Our results showed a mean V.C of 4.7 ± 1.3 and 4.7 ± 1.8 before and after laser therapy subsequently denoting moderate mitral regurgitation (3&#;5 mm) with no significant P = 0.284 as in Table .

On the basis that regurgitant area/left atrial area less than 20% considered mild, 20&#;40% moderate, and more than 40% severe; our study revealed that 20 (74%) patients were in moderate mitral regurgitation (MR), 3 (11%) patients were in severe mitral regurgitation and 4 (15%) patients were in mild regurgitation before the study, while after laser therapy, 19 (7.03%) patients were in moderate MR, 2 (7.4%) patients were in severe MR and 6 (22.2%) patients were in mild MR.

On the other hand, there was no change in diastolic function before and after laser therapy, 22 (81%) of the patients were in diastolic dysfunction grade three before LLLT, only 2 (7.4%) patients showed diastolic dysfunction grade one and 3 (11.1%) of them were in diastolic dysfunction grade two after LLLT. The change in the ratio of septal E/e was assessed and the mean was 17.0 ± 5.0 before laser therapy which became 18.0 ± 5.0 after laser therapy denoting no significant change with both measures P = 0.913 as in Table .

Regarding the six-minute walk distance, the mean was less than 200 m before the study (149 ± 39.0 m) that was changed to more than 300 after laser therapy (385.074 ± 61.740) with statistically significant P < 0.001 denoting also significant clinical improvement as in Table .

NYHA classification and six-minute walk distance were monitored before and after LLLT, regarding NYHA classification, 15 (55.5%) patients were in class III, 12 (44.4%) were in class IV, none of the patients in class II before laser therapy while after laser therapy; 25 (92.5%) patients shifted to class II, 8 (29.6%) patients were in class III with P < 0.001 denoting a statistically significant clinical improvement as shown in Table .

A total of 67 patients were assessed for eligibility during the study period. Where 37 of them were not meeting the inclusion criteria, and 3 of them were dropped from the trial because of technical issues with the laser acupuncture machine during regular sessions. In the overall sample, 21(77.8%) of the patients were males while 6 (22.2%) were female, the mean age was 57.7 ± 6.89 years. Of the 25 patients had a non-viable myocardium as per dobutamine echocardiography, and 8 patients refused revascularization. 19 (70.3%) of 27 patients were smokers, and 15 of them were diabetics for more than 10 years. 6 (22.2%) patients were hypertensive for years. Also, all patients were in sinus rhythm with a narrow QRS complex. All baseline characteristics of patients are shown in Table .

Discussion

To the best of our knowledge, the current study is the first clinical study to evaluate the clinical outcomes and echocardiographic parameters in patients with left ventricular systolic failure after low-level laser therapy, a topic that has not been thoroughly addressed in the literature.

Even though the therapeutic mechanism of LLLT on cardiac muscle is still not fully known, numerous studies have suggested that it may have potent anti-inflammatory effects in the clinical setting [18]. LLLT can suppress circulating cytokines levels, in particular, tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, these cytokines can promote progressive left ventricular (LV) dysfunction, LV remodeling, and cardiomyopathy [19]. Another explanation for how LLLT improves cardiac function is that it does by increasing the expression of vasoactive peptides and enhancing the production of nitric oxide (NO), which would reduce the acute inflammatory response in the myocardium and has a beneficial impact on LV function but at low concentrations [20, 21].

The findings of the current study showed an improvement in LV function after LLLT, which may be explained by the release of nitric oxide, which causes peripheral vasodilatation, reduces peripheral resistance, and then improves cardiac output [22]. Also, may be explained by an improvement in cardiac muscle performance due to the increased intake of energy provided by the increased aerobic metabolism stimulated by LLLT [9, 23]. Our findings were supported by several experimental studies that demonstrated the impact of low-level laser therapy on the infarction size, geometry, and LV function following myocardial infarction. These studies exhibited a decrease in infarct size and an improvement in cardiac performance [21]. Indeed, Low-level laser therapy was applied in a single, small-sample-size clinical study that reduces cardiac cellular damage and accelerates cardiac performance in patients recovering from coronary artery bypass grafting [24].

The New York Heart Association (NYHA) classification and the 6-min walking test (6MWT) have been widely supported by medical societies around the world and have been used in clinical studies showing the beneficial effects of various medications on mortality and morbidity in patients with chronic systolic failure [25]. In our study, we use the NYHA classification and the six-minute walk test to assess the clinical impact of LLL therapy in patients with chronic systolic failure. The results showed that low-level laser therapy was effective in reducing NYHA and 6-mwt. In contrast, Bublitz et al. conducted a pilot trial using LLLT on hospitalized patients with decompensated Heart Failure who then underwent a 6-min walking test, the total distance during this functional test was unaffected by 28 J of LLLT or 100 MW power [26]. However, clinical trials and a meta-analysis established that functional performance would be varied based on the population investigated and the laser dose applied [27, 28].

Unfortunately, our study found no statistically significant difference in left ventricular diastolic function. The lack of a beneficial effect of LLLT on diastolic function. Patients in this group are not a homogenous group. It's caused by a wide variety of disorders that may require individualized treatment [29] Additionally, the SGLT-2 inhibitor is currently the only type of drug or treatment that has significantly improved this group of patients [30].

It is important to note that mitral regurgitation significantly increased end-diastolic and end-systolic volumes of the left ventricle, indicating adverse cardiac remodeling and worse systolic function [31]. In this context, our study evaluated mitral regurgitation through the assessment of vena contracta width and regurgitation area to the left atrial area before and after laser therapy, but without any significant change. This may be due to the short follow-up period.

Peak tricuspid regurgitation pressure is a critical measurement for evaluating pulmonary artery systolic pressure and an important parameter in detecting the severity and follow-up of patients with left ventricular systolic heart failure [32]. Our study revealed a statistically significant difference between the mean peak TR pressure measurements before and after laser therapy; this finding was supported by a study conducted by Sayed et al. that examined the impact of LLLT in patients with chronic obstructive pulmonary disease and evaluated the echocardiographic of the right ventricular functions. It was discovered that low-level laser therapy did not lead the PASP to increase [33].The importance of NO for the cardiac function is still hotly debated, and a number of mechanisms of inotropic effects of low-level laser therapy. NO have been clarified in experimental studies. These include the cGMP-mediated inhibition of phosphodiesterase and subsequently increased cAMP [34, 35], direct activation of adenylyl cyclases [36], and enhanced excitation&#;contraction coupling by S-nitrosylation [37] or by increasing contractile calcium responsiveness [38]. Nitric oxide levels in the patients who were enrolled on our study were assessed, and it was found that they were statistically significant both before and after laser therapy. These findings were corroborated by experimental studies which showed an increase in NO levels in cardiac muscle that was associated with a cardioprotective effect [39].

Some limitations should be taken into account. To start, a small sample of participants were enrolled in our study since it was difficult for patients with persistent systolic heart failure to attend appointments and because it was challenging for them to attend physiotherapy sessions regularly. Second, we were unable to evaluate regurgitant volume using PISA and EROA measurements or assess LV systolic function with GLS analysis since our echocardiogram did not support this software. Lastly, the short duration of follow-up.

Low-Level Laser Application in the Early Myocardial ...

Low-level laser therapy (LLLT) has been targeted as a promising approach that can mitigate post-infarction cardiac remodeling. There is some interesting evidence showing that the beneficial role of the LLLT could persist long-term even after the end of the application, but it remains to be systematically evaluated. Therefore, the present study aimed to test the hypothesis that LLLT beneficial effects in the early post-infarction cardiac remodeling could remain in overt heart failure even with the disruption of irradiations. Female Wistar rats were subjected to the coronary occlusion to induce myocardial infarction or Sham operation. A single LLLT application was carried out after 60 s and 3 days post-coronary occlusion, respectively. Echocardiography was performed 3 days and at the end of the experiment (5 weeks) to evaluate cardiac function. After the last echocardiographic examination, LV hemodynamic evaluation was performed at baseline and on sudden afterload increases. Compared with the Sham group, infarcted rats showed increased systolic and diastolic internal diameter as well as a depressed shortening fraction of LV. The only benefit of the LLLT was a higher shortening fraction after 3 days of infarction. However, treated-LLLT rats show a lower shortening fraction in the 5th week of study when compared with Sham and non-irradiated rats. A worsening of cardiac function was confirmed in the hemodynamic analysis as evidenced by the higher LV end-diastolic pressure and lower +dP/dt and &#;dP/dt with five weeks of study. Cardiac functional reserve was also impaired by infarction as evidenced by an attenuated response of stroke work index and cardiac output to a sudden afterload stress, without LLLT repercussions. No significant differences were found in the myocardial expression of Akt 1 /VEGF pathway. Collectively, these findings illustrate that LLLT improves LV systolic function in the early post-infarction cardiac remodeling. However, this beneficial effect may be dependent on the maintenance of phototherapy. Long-term studies with LLLT application are needed to establish whether these effects ultimately translate into improved cardiac remodeling.

Introduction

Myocardial infarction (MI) is a major cause for heart failure (HF) development (Yancy et al., ). Data are showing that three million people are affected by MI in the USA, and more than 400.000 new cases are reported for each year. In fact, ~50% of patients will die within 5 years, and 40% die 12 months after the first HF hospitalization (Kolseth et al., ).

The acute MI triggers an adverse process known as cardiac remodeling, in which there is left ventricular (LV) dilation and enlargement of the ischemic tissue (Serra and Tucci, ). Moreover, an impaired LV systolic and diastolic function and a reduced myocardial inotropism are well-documented findings (dos Santos et al., ; Antonio et al., ). Several mechanisms are shown to be implicated in cardiac remodeling, including adrenergic hyperactivity, renin-angiotensin-aldosterone system, apoptosis, autophagy, fibrosis, inflammation, oxidative stress, calcium handling abnormalities, and metabolic dysfunction (Whelan et al., ; Carlos et al., ; Ziff et al., ). Moreover, post-infarction cardiac remodeling is associated with a higher prevalence of cardiac rupture, arrhythmias, and formation of aneurysms. In the long term, there is the development of HF and sudden death (Whelan et al., ; Ziff et al., ).

Several interventions have been proposed to alleviate cardiac remodeling to prolong or prevent the development of HF (Carlos et al., ). However, current therapies have shown only modest results in survival or potential adverse properties (Yancy et al., ; Grosman-Rimon et al., ). In latest years, experimental studies have punctuated that the low-level laser therapy (LLLT) may be a promising approach to modulate various biological processes (Albertini et al., ; Pires et al., ). The LLLT stimulates photoreceptors in the mitochondrial respiratory chain, resulting in increased ATP, increased growth factor secretion and tissue healing (Tuby et al., ; Huang et al., ; Peplow et al., ). A cardiac LLLT effect has been reported for over 10 years, in which infarcted rats showed a lower myocardial necrosis (Oron et al., b), LV dilatation (Ad and Oron, ; Yaakobi et al., ), and most favorable milieu to prevent scar disruptions (Whittaker and Patterson, ) with LLLT. More recently, our group demonstrated reduced infarct size, attenuated the systolic dysfunction and beneficial modulates inflammation and expression of vasoactive peptides in rats submitted to LLLT (Manchini et al., ).

In a recent systematic review, we have reported that many studies have only assessed the LLLT role at MI early stage, in which data reporting effects on the progression to HF are limited (Carlos et al., ). Moreover, an intriguing is issue shown to be an attenuated cardiac remodeling in animals submitted to LLLT only at the initial phase of injury. In this regard, it has been shown benefits of LLLT after several weeks post-MI, e.g., decreased infarct size and cardiac dilation (Oron et al., b; Yaakobi et al., ). Although these data indicate that the benefits of LLLT in the acute phase of MI may persist in overt HF, there are some limitations that should be considered: (i) there is no blinding for the experimental group or outcomes. A more suitable method would be to blind the infarct size and LLLT; (ii) inclusion/exclusion criteria has not been stated (e.g., animals with similar infarct sizes). The control of infarct size it seems to be a key issue because the remodeling is intensified on larger infarctions. Thus, it is doubtful to consider a beneficial cardiac remodeling LLLT effect because of the intragroup infarct size variability; (iii) there is only cross-sectional design studies, and the causality results cannot be determined. Therefore, this study was designed to determine whether LLLT application benefits at the MI early stage remains in overt HF same with disruption of treatment.

Materials and Methods

Animals and Experimental Design

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 ). The protocol was approved by the Institutional Research Ethics Committee of the Nove de Julho University, São Paulo, Brazil (number: &#;). 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., ). 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, ). 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.

FIGURE 1

Figure 1. Experimental design protocol.

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%.

MI Model

The MI was induced according to a well-established technique (Antonio et al., ). 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.

Phototherapy

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.

TABLE 1

Table 1. Protocol of LLLT irradiation.

Echocardiography

Rats were anesthetized as described above (K-X mixture) and LV echocardiography was performed using a 12-MHz transducer connected to an HP Sonos- (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., ). 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., ). Diastolic function was not evaluated owing to the fusion of the A and E waves.

LV Hemodynamic Study and Afterload Stress

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., ). Thereafter, sudden LV afterload increases were achieved using a single phenylephrine in bolus injection (15&#;25 μg/kg, i.v.) (dos Santos et al., ).

Biometric Data

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

Myocardial Fibrosis

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).

Western Blot

Proteins were extracted from the LV remote area as previously described by us (Silva et al., ). 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: dilution; Abcam, Cambridge, MA, USA); rabbit anti-phosphoSer473Akt1 (1: dilution; Abcam, Cambridge, USA); goat anti-VEGF (1:; 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:; 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).

Statistical Analysis

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.

Results

LLLT Does Not Affect Structural and Functional Abnormalities of LV

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.

TABLE 2

Table 2. Biometric data.

FIGURE 2

Figure 2. Representative microphotographs of remote myocardium with 3 days (A: Sham; B: MI-N; C: MI+LLLT) and 5 weeks (D: Sham; E: MI-N; F: MI+LLLT) after MI. Myocardial sections were stained with Masson's trichome. (G) Is representative of the statistical comparisons between experimental groups. Data are means ± SD (n = 4 per group). P-values were determined by one-way ANOVA and post hoc Newman-Keuls test. Magnification 40x (scale bar: 50 μm). #p < 0.05 vs. 3 days.

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.

FIGURE 3

Figure 3. Effects of LLLT in the ventricular cavity and fractional shortening 3 days and 5 weeks after MI (Sham, n = 8; MI-N, n = 7; MI+LLLT, n = 6). Compared with the Sham group, the left ventricular end diastolic diameter (LVDd) were significantly augmented in 3 days (MI+LLLT group) and 5 weeks (MI-N and MI+LLLT). The left ventricular end systolic diameter (LVSd) was higher in all infarcted groups compared with Sham group (3 days and 5 weeks). The fractional shortening of the MI-LLLT group was significantly higher than MI-N group on 3 day, however MI+LLLT showed significantly lower LV performance in 5 weeks when compared with Sham and MI-N groups. Data are means ± SD. P-values were determined by two-way repeated ANOVA complemented by Bonferroni post-hoc. #p < 0.05 vs. 3 days.

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., ). 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.

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FIGURE 4

Figure 4. Repercussion on baseline hemodynamic and sudden afterload after 5 weeks of MI (Sham, n = 8; MI-N, n = 7; MI+LLLT, n = 6). Data are means ± SD. P-values were determined by one-way ANOVA and post hoc Newman-Keuls test. LVEDP: left ventricular end-diastolic pressure; +dP/dt: maximum positive time derivative of developed pressure; &#;dP/dt: maximum negative derivative of developed pressure; SWI: stroke work index CO: cardiac output.

Survival/Angiogenesis Factors Are Not Affected by MI or LLLT

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., , ). 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.

FIGURE 5

Figure 5. The protein expression by western blot in the remote myocardium with 3 days and 5 weeks after MI. (A,E) Protein expression of Akt1. (B,F) Protein expression of pAkt1. (C,G) Ratio of pAkt1/ Akt1. (D,H) Protein expression of VEGF. All values were normalized to levels of GAPDH (Sham, n = 8; MI-N, n = 7; MI+LLLT, n = 6). Data are means ± SD. P-values were determined by one-way ANOVA and post hoc Newman-Keuls test.

Discussion

Data showing that LLLT application only at the MI early stage could result in a long-term beneficial effect on cardiac remodeling are intriguing. In fact, LLLT action has been achieved until several weeks after discontinuing of the irradiation, e.g., a minor infarct size (Oron et al., b).

We showed here that the LLLT improved LV systolic function only 3 days post-infarction, which confirms previous data from our lab (Manchini et al., ). On the other hand, we have not reported a cardioprotective LLLT role during evolution to overt HF, as illustrated by the no effect on infarct size, cavity dilation and LV systolic performance at the end of the study. Yang et al. () have published similar findings in rats subjected to LLLT with up to 72 h post-infarction. Accordingly our data, these authors also reported no beneficial LLLT effect in LV diastolic and systolic diameter as well as LV performance on echocardiographic analysis. We advance these findings to explore whether the LLLT could increase functional heart reserve for an increased LV afterload. In fact, a minor functional LV reserve shown be a marker for cardiac remodeling progression (Fletcher et al., ), in which it can be the result of a decreased myocardial inotropism at a given loading level (Francis et al., ). As illustrated in Figure 3, infarcted rats had exacerbated LVEDP and decreased +dP/dt, &#;dP/dt, SW, and CO as a response to sudden afterload increases. Mechanisms associated with changes in cardiac performance are not fully clarified, but they may be linked to an altered handling Ca2+ and myofilament Ca2+ sensitivity (Pfeffer and Braunwald, ). Moreover, post-infarct ventricular dilatation is shown to be limiting for the intracavitary pressure development, as defined by the Laplace (Pfeffer and Braunwald, ; dos Santos et al., ). Importantly, LLLT had no effect on the functional cardiac abnormalities.

To our knowledge, infarct size has been the main variable affected by LLLT, and many studies have shown a minor injury size with several weeks post-LLLT application (Oron et al., a,b; Yaakobi et al., ; Yang et al., ). It is hard to understand the differences of our findings to previously studies. A key reason may be the randomization, in which we have only included animals with large infarcts. The comparison of experimental groups that have a similar infarct size at baseline is a critical issue to avoid the causality of results and has not been controlled in previous studies. Thus, while it may be understood that the LLLT lead to a lower infarct size, it is possible also that rats with lower infarct sizes have been included in the LLLT-treated group. Moreover, previous investigations have only carried out a cross-sectional analysis (Oron et al., a; Yaakobi et al., ; Yang et al., ), in which the causality cannot be determined. In this regard, we have analyzed the longitudinal repercussion of LLLT (Table 2) to clarify whether infarct size at baseline (3 days) changed over time as an effect of phototherapy. Other issues that show be investigated to understand the differences in our findings for previously studies are (i) the differences in irradiation parameters and (ii) approach to analyzing the infarct size (e.g., histomorphometric or echocardiographic).

Cardioprotective effects of LLLT are often attributed to angiogenic factors in a wide range of tissues (Dourado et al., ; Feng et al., ; Cury et al., ), including the ischemic myocardium (Tuby et al., ). Thus, there are findings showing greater pro-angiogenic stimuli (e.g., VEGF expression) in infarcted hearts that received LLLT only in the early MI (Mirsky et al., ; Zhang et al., ). In our study, there was no increased VEGF expression and its well-known downstream&#;Akt with 3 days and 5 weeks post-MI. It is shown to be reported that time of analysis of VEGF post-infarction may be a reason for our findings. Zhao et al. () conducted experiments on infarcted rats to investigate the temporal expression of angiogenic factors. The authors observed a significant increase in the VEGF protein levels at the border zone only during day one post-MI and with subsequent decline in 28 days. Consequently, we cannot exclude an effect of LLLT on angiogenic VEGF signaling because our analysis may have been influenced by the timeline.

In summary, our findings illustrate that LLLT improves LV systolic function in the early post-infarction cardiac remodeling. However, this beneficial effect may be dependent on the maintenance of phototherapy. Long-term studies with LLLT application are required to establish whether these effects ultimately translate into improved cardiac remodeling.

Author Contributions

MM, drafted the work and substantially contributed to work design, as well as, acquired, analyzed and interpreted all data. EA, drafted the work and substantially contributed to work design, as well as, acquired, analyzed and interpreted all data. JS, drafted the work and substantially contributed to work design, as well as, acquired, analyzed and interpreted the all data s and protein expression protocols. Pd, laser protocol and dosage. RA, laser protocol and dosage. FP, drafted the work and substantially contributed to work design, as well as, acquired, analyzed and interpreted all data. RF, performed experiments and protein expression protocols. JM, Echocardiogram analysis. SV, drafted the work and substantially contributed to work design, as well as, acquired, analyzed and interpreted all data. VG, drafted the work and substantially contributed to work design, as well as, acquired, analyzed and interpreted all data. Md, performed experiments and protein expression protocols. AY, performed experiments and histological analysis. MC, performed experiments and histological analysis. Rd, drafted the work and substantially contributed to work design, as well as, acquired, analyzed and interpreted all data. DB, performed experiments and protein expression protocols. Bd, performed experiments and protein expression protocols. PT, oversaw the design and performance of the experiments, analyzed data, interpreted the results of the experiments and edited the final format of the manuscript. AS, oversaw the design and performance of the experiments, analyzed data, interpreted the results of the experiments, edited and revised manuscript. All authors revised the work critically, approved the final version to be published and declared accountable for all aspects of the work.

Funding

This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (grant #/-8) and FAPESP (Grants: 09-/8; 15/-9).

Conflict of Interest Statement

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.

Abbreviations

MI, Myocardial infarction; HF, Heart failure; LLLT, Low-level laser therapy; LV, Left ventricular; ATP, Adenosine triphosphate; NHI, National Institutes of Health; MI-LLLT, Infarcted rats submitted with laser; MI-N, Infarcted rats non-treated with laser; AlGaInP, Aluminum Indium Gallium Phosphorus; EDP, End diastolic pressure; +dP/dt, Positive derivatives of the developed pressure; -dP/dt, Negative derivatives of the developed pressure; SV, Stroke volume; SP, Systolic pressure; SWI, Stroke work index; CO, Cardiac output; VEGF, Vascular endothelial grow factor.

References

Ad, N., and Oron, U. (). Impact of low level laser irradiation on infarct size in the rat following myocardial infarction. Int. J. Cardiol. 80, 109&#;116. doi: 10./S-(01)-4