Ultrastructure Of Injured Mitochondria Induced By Ca²⁺ And Oxygen-free Radicals

As the energy producer of the Eukaryotic cells, in addition to supply energy,mitochondria are also involved in cell differentiation, cell communication and cell apoptosisand play an important role in the regulation of cell growth and cell cycle. So in recent years,researches on how to control mitochondrial damage to control mitochondria related diseasesare more and more. This article aims to study on the ultrastructural changes of mitochondriato explore the mechanism of injury isolated mitochondria from the swine heart induced byCa2+and oxygen free radicals, and provides some data to the researches about ischemicheart disease, cardiac hypertrophy and some other mitochondrial damage related heartdisease. We added10~200μmol/L of CaCl2and0.5~5μmol/L of H2O2respectively toinduce the mitochondria injury， than we get some pictures of the internal ultrastructureof mitochondria by transmission electron microscopy (TEM), and scan the externalmorphology of them by the atomic force microscope (AFM) to determine the damage fromCa2+and oxygen free radicals on the isolated pig heart mitochondria. It turns out that in theconcentration range in our experiment, with the concentrations of CaCl2and H2O2increasing, the proportion of damaged mitochondria increased, the flameng score becamehigher, and that means the mitochondria damaged more. High concentrations of Ca2+andoxygen free radicals can lead to the splitting of mitochondrial crest, collapse ofmitochondrial membrane damage, changes in the matrix, and the differences are obvious.From the observation of the different damaged stages of the mitochondrial ultra structure bytransmission electron microscopy，we got the flameng damage model of the process ofmitochondrial destroyed, and by the mitochondrial statistical injury score in differentdamage stages, the relationship of concentrations of CaCl2and H2O2and the mitochondrialintegrity is obtained. We scan the mitochondria in tapping mode of the AFM, and got somemore clear and reliable mitochondria photos than the previous studies, and comparing to theresult from TEM we obtained the corresponding results of mitochondrial5stages damagemodel. Level0: the surface structure is intact, smooth and slightly wrinkled; the crosssection is semicircular and shows a slight fluctuation; the height is of about260nm. Level1:the structural of the surface is integrity; Wrinkles on the surface deepened, the height islower slightly, the cross-section is similar to trapezoid that means a substantially flat surfacewith more significant fluctuations, but there are no significant collapse phenomenon; Level 2, surface structural is integrity, showed apparent collapse, they were generally lower in themiddle and higher on the sides,some mitochondria showed partial collapse on one headbut integrity one the other one; Level3, the surface structure showed a complete collapsevery obviously, cross-section is like a trapezoid again，but the overall height was droppedto about100nm, at this stage there are some obvious swelling mitochondria, smoothsemicircular cross-sectional view presented, but the height is about400nm; Level4, themitochondria are overall collapse, merge into an oval shape spreading film, with theparticle distribution on the surface，and the height is about50nm.And we have statistics theaverage sizes and heights of different stages of damaged mitochondria, obtained relationsbetween H2O2and CaCl2concentration and external morphology of the damagedmitochondria. These results will provide a reliable basis for the future mitochondrialdamage experiments by atomic force microscopy. In addition, we also found thatmitochondria divided due to the irritation for injury protection, will be corroborate to thephenomenon that mitochondria adapt to the new environment of chronic injury by theproliferation.