Low Energy Visible Light Induces Reactive Oxygen Species Generation and Stimulates an Increase of Intracellular Calcium Concentration in Cardiac Cells

Low energy visible light (LEVL) irradiation has been shown to exert some beneficial effects on various cell cultures. For example, it increases the fertilizing capability of sperm cells, promotes cell proliferation, induces sprouting of neurons, and more. To learn about the mechanism of photobiostimulation, we studied the relationship between increased intracellular calcium ([Ca2+]i) and reactive oxygen species production following LEVL illumination of cardiomyocytes. We found that visible light causes the production of O2. and H2O2 and that exogenously added H2O2 (12 microm) can mimic the effect of LEVL (3.6 J/cm2) to induce a slow and transient increase in [Ca2+]i. This [Ca2+]i elevation can be reduced by verapamil, a voltage-dependent calcium channel inhibitor. The kinetics of [Ca2+]i elevation and morphologic damage following light or addition of H2O2 were found to be dose-dependent. For example, LEVL, 3.6 J/cm2, which induced a transient increase in [Ca2+]i, did not cause any cell damage, whereas visible light at 12 J/cm2 induced a linear increase in [Ca2+]i and damaged the cells. The linear increase in [Ca2+]i resulting from high energy doses of light could be attenuated into a non-linear small rise in [Ca2+]i by the presence of extracellular catalase during illumination. We suggest that the different kinetics of [Ca2+]i elevation following various light irradiation or H2O2 treatment represents correspondingly different adaptation levels to oxidative stress. The adaptive response of the cells to LEVL represented by the transient increase in [Ca2+]i can explain LEVL beneficial effects.


Introduction
Life on earth is entirely dependent upon the interaction of sunlight with cells especially in plant photosynthesis (1). Sunlight also has medical benefits, which have been exploited for over thousands of years in ancient Egypt, India and China in treating skin diseases, psoriasis, vitiligo and even cancer (2). Recent observations show that even low energy visible light (LEVL) can serve as a medical tool. For example, LEVL increases the rate of wound healing (3), enhances the fertilizing capability of sperm cells (4) and increases the rate of healing bone defects (5). In vitro studies have found that LEVL increases proliferation of cells as fibroblasts (6), keratinocytes (7), and lymphocytes (8), and induces the respiratory burst in neutrophils (9). The mechanism of photobiostimulation by LEVL is still unclear. It has been suggested that reactive oxygen species (ROS), which can be produced by photosensitization of endogenous cell chromophores such as cytochromes (10), flavins/riboflavins (11) and NADPH (12), may have an important role in this light/tissue interaction (13)(14)(15). The suggestion is based on the recent recognition that small amounts of ROS are considered to be important for mediating cell activities (16)(17)(18)(19). The production of ROS in response to low energy visible light has been demonstrated in fibroblasts (20), sperms (21) and lymphocytes (15). In addition, it has been found that LEVL causes [Ca 2+ ] i elevation in cells like sperm (4) and skin (22). factors and hormones were shown to stimulate ROS production, which were dependent on [Ca 2+ ] i rise (27). The relationship between ROS and [Ca 2+ ] i has been suggested to involve the redox-sensitive transcription factor N kappa β, which was found to change [Ca 2+ ] i homeostasis in response to changes in the redox state of thiol groups (28). The kinetics which characterizes the [Ca 2+ ] i elevation has been shown to be an important parameter determining the kind of signal which will be evoked. The microscope was focused on a single cardiomyocyte or a group of two to three cells in the indo-1 loaded culture. Every 2-5 minutes the average fluorescence was measured for 10 seconds by using software written by D. Kaplan, Biological Institute

Illumination and H 2 O 2 treatment
While on the chamber of the microscope, the cells were irradiated from

Involvement of L-type calcium channel
To determine whether changes in intracellular calcium are mediated by a specific calcium-channel, 10 µM of verapamil (L-type voltage-dependent calcium channel blocker) were added to cardiac cells before LEVL irradiation or H 2 O 2 treatment and then the indo-1 fluorescence was measured. nor did production of O 2 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) respectively (these data will not be presented).

Structural changes following illumination or H 2 O 2 treatment
Myocytes in PBS were treated with increasing concentrations of hydrogen peroxide or were illuminated with different doses of light. After 50 min, the cells were returned to growth medium and placed in a 5% CO 2 incubator environment at 37°C.

Lactate dehydrogenese (LDH) assay
Cytotoxicity was assessed by activity of released LDH into the culture medium.
The LDH activity was measured using an LDH kit as described before (38). The results are expressed as percent of LDH released in samples relative to samples in which cells were lysed with 1% Triton x-100.

ROS production by cardiomyocytes
In order to verify the generation of ROS in response to visible light we measured OOH is shown in the detectable range. Illuminating cardiomyocytes while scanning the EPR spectrum for 83 seconds, resulted in the appearance of a spectrum which is compatible with that of DEPMPO-OOH (Fig. 2b), while the non-illuminated cell suspension spectrum exhibited only background noise (Fig. 2c). Addition of superoxide dismutase (SOD), a superoxide oxide anion scavenger, to the cell suspension decreased the intensity of the DEPMPO-OOH spin adduct signal (Fig. 2d).
These results show that LEVL illumination increases the level of O 2 •in illuminated cardiomyocytes.

Effect of visible light irradiation on [Ca 2+ ] i
We next determined whether light could directly induce increased intracellular [Ca 2+ ] i even without exogenous photosensitizers. We found that illumination at an energy density of 3.6 J/cm 2 cause a semi-transient elevation of [Ca 2+ ] i , with a broad peak of 12% which lasted for more than 30 min followed by a decrease to a stable plateau of 8% above the control (Figs. 3b, 7a). Increasing the light energy density to 12 J/cm 2 resulted in a linear elevation of [Ca 2+ ] i , which reached 25%, 60 minutes after illumination (Fig. 3c). Nevertheless, this elevation was reduced to 7 % above control, as a result of including 200 U/ml catalase (H 2 O 2 scavenger) into the medium before illumination, (Fig. 3d). In the control, the observed [Ca 2+ ] i values fluctuated up to 3 % above and 2 % below the basal level during 80 minutes (Fig. 3a).

Effect of H 2 O 2 on [Ca 2+ ] i
To support our hypothesis that ROS take part in the pathway leading to the

Effect of light or H 2 O 2 on the viability of the cells
To rule out the possibility that visible light, at the doses employed, causes damage to the cell membrane, we measured LDH secretion to the medium immediately, 2.5, 6 and 24 hours after 3.6 or 12 J/cm 2 visible light illumination (LDH is a biochemical marker for the integrity of the membrane (39)). We found that the amount of LDH released to the medium at various times after 3.6 J/cm 2 or 12 J/cm 2 illumination was similar to that of the LDH released in non-illuminated cultures ( Fig. 5) and the increase in LDH level during 24 hours is only due to natural exocytotic release.
Another approach for determining the effect of light or H 2 O 2 , was to observe the cell morphology 24 hours after illumination or treatment with H 2 O 2 (Fig. 6). The cultured cells were immunocytochemically stained for α-sarcomeric actin to observe the contractile filaments and were counterstained with hematoxylin to observe the nucleus. In the control (Fig. 6A), most of the cardiomyocytes were flattened with strands of well-organized myofibrils α-sarcomeric actin with evident cross-striation.
The nucleus showed a well stained chromatin structure. Treatment with 12 µM of structure and a slight loss of α-sarcomeric actin staining. No changes were shown in the nucleus (Fig. 6B). Increasing the H 2 O 2 concentration to 24 µM caused focal disorganization of myofibril structures, vacuolization of the cytoplasm (blue arrow) and picnotic damage to many nuclei (white arrow). Nevertheless, approx. 65% of cells did not exhibit changes (Fig. 6C). By further increasing the H 2 O 2 concentration to 48 µM, a severe alteration of the α-sarcomeric actin positive structure, disorganization of the myofibrils, vacuolization of the cytoplasm and perinuclear edema (blue arrow) was induced. The nucleus showed picnotic damage (white arrow) (Fig. 6D).
Illumination at 3.6 J/cm 2 caused no visible alteration in cardiomyocyte structure, as seen in stained α-sarcomeric actin myofibrils and maintenance of cross-striation ( Fig. 6E). Increasing the illuminating energy to 7.2 J/cm 2 caused a decrease in the α-sarcomeric actin staining, but the nucleus was without visible changes (Fig. 6F).
Increasing the illumination energy to 12 J/cm 2 caused a disorganization of the myofibril structures, decrease in α-sarcomeric actin staining, vacuolization of the cytoplasm and oncotic damage (swelling) of the cytoplasm, though no visible damage to the nuclei was seen (Fig 6G). Adding exogenous catalase (200 U/ml) to the medium of cardiomyocytes culture before irradiation with 12 J/cm 2 protected the cells, as seen in Figure 5H.  (Fig 3c) or a semi-exponential increase, (Fig 4c) (Fig. 3d), which also prevented cell damage (Fig. 6H)

Light-induced [Ca 2+ ] i elevation and its linkage to ROS
It is shown here that visible light at the energy density of 3.6 J/cm 2 (60). Moreover, it has been shown that the mass content of IP 3 is lower in isolated cells (which were used in this study) than in the intact tissue (61). Therefore, the IP 3 contribution to the overall calcium homeostasis in cultured cells appears to be negligible (62).