Original articleNear infrared light protects cardiomyocytes from hypoxia and reoxygenation injury by a nitric oxide dependent mechanism☆
Introduction
Light in the red to near infrared (NIR) range (630–1000 nm) generated by using low energy laser or light-emitting diode (LED) arrays has been reported to have beneficial biological effects in many injury models. Such photobiomodulation has been observed to increase mitochondrial metabolism [1], [2], [3], [4], facilitate wound healing [5], [6], [7] and promote angiogenesis in skin [5], bone [8], nerve [9] and skeletal muscle [10], [11], [12], [13]. Despite its widespread therapeutic potential, the mechanisms responsible for the therapeutic actions of photobiomodulation by NIR have not been elucidated in detail.
Interestingly, beneficial effects of NIR were frequently observed in injuries caused after metabolically challenging mitochondria. NIR reversed the toxic effects of tetrodotoxin (TTX), a voltage-dependent sodium channel blocker and down regulator of cytochrome c oxidase (COX), and potassium cyanide (KCN), an irreversible inhibitor of COX, in primary neurons [6], [14]. NIR improved retinal function in an animal model of mitochondrial dysfunction caused by methanol-induced formate, a reversible COX inhibitor [15]. These in vitro and in vivo studies suggest that modulation of mitochondrial proteins having chromophore-containing groups such as COX plays an important role in photobiomodulation, particularly under conditions when mitochondria are metabolically challenged. On one hand, mitochondria have been recognized central to the development of ischemic injury. They play a role in apoptotic cell death, by releasing pro-apoptotic factors into the cytoplasm which activate caspases [16]. Apoptosis as well as necrosis contribute to tissue injury in myocardium following ischemia and reperfusion. Between 5 and 30% of cardiomyocytes undergo apoptosis in the rodent and human heart within 16 h of reperfusion [17], [18], [19] and this trend persists for months [19], [20]. On the other hand, mitochondria are potential sites for protection of the heart and other organs, as evidenced by their importance in ischemic or pharmacologic preconditioning and postconditioning [21], [22], [23].
In vivo studies have demonstrated protection of myocardium from ischemic injury by NIR. In an experimental model of myocardial infarction, Oron et al. showed a profound effect of repetitive exposure of chronic infarcted myocardium in rats and dogs to low energy lasers (803 nm), resulting in a 50–70% reduction in infarct size 4–6 weeks after left descending coronary artery occlusion [24]. An upregulation in the expression of inducible nitric oxide synthase (NOS) and vascular endothelial growth factor was associated with cardioprotection and enhanced angiogenesis [25]. In most previous investigations with NIR treatments, light exposure was applied repeatedly over a relatively long time frame before and/or after stress causing injury. Similarities observed between the effects of postconditioning and NIR exposure encouraged us to test NIR treatments using protocols designed for postconditioning experiments. Interestingly, in an open-chest rabbit model, NIR (670 nm) provided powerful cardioprotection against ischemia and reperfusion injury when myocardium was exposed for 5 min at the onset of reperfusion. (P. Pratt, personal communication, a manuscript on these results is under submission). The goal of the present investigation was to characterize the effect of NIR on cultured rat and mouse cardiomyocytes undergoing hypoxia and reoxygenation, and establish the mechanism for protection of NIR against injury without confounding factors present in vivo.
Section snippets
Materials and methods
All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of the Medical College of Wisconsin, Milwaukee, Wisconsin. All conformed to the Guiding Principles in the Care and Use of Animals of the American Physiologic Society [26] and were in accordance with the Guide for the Care and Use of Laboratory Animals [27].
NIR decreases HR-induced cell death in rat neonatal cardiomyocytes and HL-1 cells
The release of cytosolic LDH is widely used as a marker of cell membrane injury. In rat neonatal cardiomyocytes NIR was administered after hypoxia in three incremental doses. HR produced a significant increase of LDH release into the cell culture medium (304 ± 20% as compared to the normoxic control values). Following the treatment with NIR, there was a significant reduction in LDH release at the power intensity of 25 mW/cm2 (total energy density of 7.5 J/cm2; 215 ± 23%), while energy densities of
Discussion
Recently, there has been increased interest in the diverse therapeutic effects of NIR [38], but the mechanisms for this photobiomodulation are not well understood. The present investigation characterizes mechanisms of NIR that may be of practical therapeutic benefit in the treatment of coronary artery disease. We demonstrated in cultured cardiomyocytes that exposure to NIR at the onset of reoxygenation following hypoxia enhances cell survival and attenuates apoptosis. Wavelength and energy
Conflict of interest statement
The authors have no conflicts of interest pursuant to the current work.
Acknowledgments
The authors thank Ms. Chiaki Kwok, MS (Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI) and Stephanie K. Gruenloh (Department of Pulmonary and Critical Care Medicine, Medical College of Wisconsin, Milwaukee, WI) for technical assistance.
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Financial support: This study was supported in part by NIH (HL069996 (MM), HL49294 (ERJ), HL68627 (ERJ), HL054820 (DCW), GM066730 (DCW), and the Department of Anesthesiology, Medical College of Wisconsin.