Nicotinamide Pretreatment Protects Cardiomyocyte Against Hypoxia-induced Cell Death By Improving Mitochondrial Stress

Myocardial ischemia occurred at early stage after severe burn has been much focusedin fundamental research and clinical therapy. The common condition of myocardialischemia in post burn is a slow starvation of oxygen and other nutrients leading to death ofan affected cardiac muscle, and increasing evidence suggests thatischemia/hypoxia-induced cell necrosis and apoptosis are major contributors to cardiacdysfunction in post burn. It is well recognized that mitochondrial dysfunction plays acrucial role in the pathogenesis of cell death induced by myocardial ischemia.Ischemia/hypoxia often leads to excessive generation of reactive oxygen species （ROS） andloss of intracellular adenosine triphosphate （ATP） and mitochondrial membrane potential,which in turn causes mitochondrial permeability transition and release of pro-apoptoticproteins, which initiate an apoptotic program. In addition, serious or prolonged periods ofischemia could lead to cell necrosis due to the depletion of ATP. These studies raise theimportance of preserving mitochondrial function in limiting myocardial damage in postburn. Therefore，it is the key to cure cardiac muscle damage in post burn that findingpotential drug.Nicotinamide, also known as niacinamide and nicotinic acid amide, is a water-solublevitamin （vitamin B3） and is the precursor for the coenzyme β-nicotinamide adeninedinucleotide （NAD⁺）. Nicotinamide is considered to play important roles in keeping normalcellular function and metabolism. It has been found that treatment with nicotinamideimproves neurological outcome and reduces infarct volume after transient focal cerebralischemia in Wistar rats, and nicotinamide also can reduce acute cortical neuronal death andedema in traumatically-injured brains. These studies indicate the protective role ofnicotinamide against ischemia and hypoxia. Recent experimental work also suggests thatnicotinamide-rich diets protect the heart from ischemia reperfusion in mice by overexpressing sulfonylurea receptor. However, no studies have examined the direct effectsof nicotinamide pretreatment on hypoxia-induced cardiomyote necrosis and apoptosis andthe molecular mechanisms involved.In the present study, we show that nicotinamide plays a protective role in reducingcardiomyocyte necrosis and apoptosis under hypoxic conditions by inhibiting oxidativestress and improving energy production.I. Materials and methodsIn vitro experiments were adopted in the present study.Investigation on the effect of nicotinamide on cardiomyocytes agains cell death undernormoxic conditionsPrimary rat neonatal cardiomyocytes were isolated and cultured from the ventricles ofneonatal Sprague-Dawley rats （2–3days old） according to the protocols. Cardiomyocyteswere pretreated with2.5,5,10, or20mM nicotinamide for16h to determine thedose-effect relationship under normoxic conditions. Cell death was accessed by examiningprotein expression of RIP and CC3with western blot. LDH release, ATP, NAD⁺andNADP⁺content were detected after10mM nicotinamide pretreatment for16h to evaluatethe protective effects on cardiomyocytes.Investigation on the effect of nicotinamide on cardiomyocytes againsthypoxia-induced cell deathCardiomyocytes were pretreated with2.5,5,10, or20mM nicotinamide or withoutnicotinamide for16h before exposure to hypoxic conditions for6h（94%N₂，5%CO₂,1%O₂）, and then the effects on cell death were determined.Cell death was accessed byexamining protein expression of RIP and CC3with western blot. LDH release and TUNELexperiment was also used to detect cell death after10mM nicotinamide pretreatment orwithout for16h before exposure to hypoxic conditions for6h.Investigation on the protective mechanisms of nicotinamide on cardiomyocytes againsthypoxia-induced cell deathAll experiments were performed with10mM nicotinamide pretreatment for16hbefore exposure to hypoxic conditions for6h, compared to control group and hypoxiagroup. ROS content was detected with fluorescence and anti-oxidant enzymes （SOD andcatalase） with western blot, to observe the anti-oxidant effect of nicotinamide on hypoxic cardiomyocytes. ATP, NAD⁺and NADP⁺content were used to detect energy production,and mitochondrial membrane potential was used to evaluate mitochondrial homeostasis.II. Results and conclusions1. Nicotinamide reduced cell death of cardiomyocytes under normoxic conditions in adose-dependent manner （cell death was accessed by examining protein expression of RIPand CC3）, but treatment of cardiomyocytes with10mM nicotinamide for16h had no effecton LDH release and ATP concentrations. Moreover, it significantly increased intracellularNAD⁺and NADP⁺levels. These data suggest that nicotinamide reduced cell death innormoxic cardiomyocytes mainly by supplying sufficient precursor for ATP synthesis.2. Nicotinamide reduced cell death of cardiomyocytes under hypoxic conditions in adose-dependent manner.10mM nicotinamide pretreatment significantly attenuatedhypoxia-induced LDH release and decreased the numbers of TUNEL-positive cellscompared with the hypoxia group. These results demonstrated that nicotinamidepretreatment protected cardiomyocytes from hypoxia-induced cell death.3. Nicotinamide pretreatment significantly reduced ROS contents and increased theprotein levels of SOD and catalase compared with hypoxic cells, indicating thatnicotinamide pretreatment effectively reduced hypoxia-induced mitochondrial oxidativestress by increasing the anti-oxidative potential in cardiomyocytes.4. Nicotinamide pretreatment increased NAD⁺and ATP concentrations andmitochondrial membrane potential nearly to control levels, which were significantlydecreased by hypoxia treatment. In all, nicotinamide pretreatment effectively prevented thecollapse of mitochondrial membrane potential mainly by promoting energy synthesis duringhypoxia treatment.