Dukhinova M.S., Lipina T.V., PhD,

Pogodina L.S., PhD

Lomonosov Moscow State University, Russia

Analysis of age-dependent changes in myocardium of Japanese quail

BACKGROUND

Cardiovascular diseases are the leading reason of human death all over the world (2, 4). Heart aging – one of the main factors in their developing – is rather well studied only in mammals. Birds are often used in embryonic development research but there is lack of information about their aging (1). Mammalian and avian hearts have similar anatomy structure but avian heart is bigger due to body weight (3). It is an obvious advantage of the model. Moreover, avian cardiomyocytes constantly undergo the mechanical stress because of their faster heart rate (7). Japanese quails, Coturnix japonica, are also characterized by natural accelerated aging and can become one of the promising model objects in gerontology.

MATERIALS AND METHODS                   

Japanese quails (males and females) 9-12 (young) and 48-52 (old) weeks old (from Emanuel Institute of Biochemical Physics RAS) were used in our research. The left-ventricle myocardium’s tissue samples were prepared for light and transmission electron microscopy studying. The cardiomyocyte size, number of blood capillaries per one cardiomyocyte and apoptotic death level (by Tunel method) were analyzed at thick sections. Volume of myofibrils, mitochondria and lipid drops, number of long mitochondria and intermitochondrial contacts (criteria of functional heart muscle overload, (5)) were measured using electron-microscopy method. The data were analyzed by Statistica 5.5 (Mann-Whitney nonparametric U-test, ð˂0,05).

RESULTS AND DISCUSSION

Light-microscopic analysis revealed that Japanese quail’s cardiomyocytes are long narrow cells about 3 µm in diameter with thin sheets of connective tissue between them. The number of blood capillaries per one cardiomyocyte is 0,86.   These parameters were at the same level in birds of both analyzed age groups.

Although no evidence of hypertrophic changes was found during light-microscopic research, the level of apoptotic cell death is increasing in two times during the studied period of quail life. The influence of cardiomyocytes’ shortage can be already seen on electron-microscopy level.

Electron-microscopic analysis revealed the nucleus of Japanese quail’s cardiomyocyte is situated in center and has elongate form and invaginated borders. The main part of cytoplasm (up to 70%) is occupied by myofibrils that organized in parallel rows. In old quails’ cardiomyocytes this type of myofibrils’ organization is sometimes damaged. From 9 to 52 weeks of life myofibrils volume is significantly decreased. However, the particular zones where contractile apparatus is hypertrophied can be seen in cells of old birds.

Picture 1. Cardiomyocyte of 9 weeks old Japanese quail

Mitochondria are localized between myofibrilar rows and in perinuclear region; their volume is 30% of cytoplasm at the average. The mitochondria’s volume is significantly increased in the cells of 52 weeks old birds. So, the mitochondria/myofibrils ratio is shifted to mitochondria in old birds’ heart, comparing with younger ones.

Due to localization, mitochondria are divided into two groups, or populations: interfibrilar and perinuclear mitochondria. These organells differ by its size and shape. Perinuclear mitochondria are mostly not very big, round- or oval-shaped organells. In interfibrilar zone long and large ones can be seen, but not very often. We account mitochondrion as long if it is more than 2 µm in length. It can be clearly seen that large mitochondria are formed by fusion of the usual organells.

Picture 2. Cardiomyocyte of 52 weeks old Japanese quail

The senescence-connected structural and functional alterations of mitochondria can be seen on electron-microscopy level. First of all, the number density of long mitochondria increased by 60% from 9 to 52 weeks age. The longest mitochondrion’s profile measured in young cardiomyocytes is 3,3 µm and runs to 6,6 µm in the old ones.

Age-related changes have different influence on number of intermitochondrial contacts in interfibrilar and perinuclear zones. No changes are marked in the interfibrilar population of mitochondria. At the same time in the perinuclear zone we can see the increase of intermitochondrial contacts’ number from 9 to 12 weeks and decrease of its quality to 52 weeks of age back to the level of 9 week-old group.

Both in interfibrilar and perinuclear zones lipid drops and lipofuscin granules can be seen.

Lipofuscin granules can be detected in cardiomyocytes of young and old quails, but its quality significantly increases in old birds. This fact corresponds well to data mentioned earlier for aging in humans (8) and in rodents (6). Lipofuscin granules in quail’s cardiomyocytes are heterogeneous, electron-dense structures of different shape and size.

The volume of lipid drops increases on 40% during studied period of life. This fact, together with mentioned above mitochondria changes, are likely to indicate alterations in cell energy metabolism during quail life.

So, we found that quail myocardium of 48-52 weeks old birds (old), in comparison with 9-12 weeks old, have several differences: increased number of apoptotic cells; increased mitochondria/myofibril ratio, increased number of long mitochondria, higher level of lipid drops volume and lipofuscin granules quality in cardiomyocytes of old heart. All mentioned changes were the same for males and females - we found no sexual distinctions in aging process in quail myocardium.

ACKNOWLEDGMENTS

We thank Dr. Pavel P. Zak and employees of Laboratory of Photo-Chemical Bases of Reception, Emanuel Institute of Biochemical Physics RAS, for provided research material and assistance in experiments realization.

REFERENCES 

1.     Henk P. J. Buermans, Bram van Wijk, Margriet A. Hulsker, Niels C. H. Smit, Johan T. den Dunnen, Gertjan B. van Ommen, Antoon F. Moorman, Maurice J. van den Hoff, Peter A. C. ’t Hoen. Comprehensive Gene-Expression Survey Identifies Wif1 as a Modulator of Cardiomyocyte Differentiation. PLoS ONE | www.plosone.org December 2010 | Volume 5 | Issue 12 | e15504

2.     Dao-Fu Dai and Peter S. Rabinovitch. Cardiac Aging in Mice and Humans: the Role of Mitochondrial Oxidative Stress. Trends Cardiovasc Med. 2009 October; 19(7): 213–220. doi:10.1016/j.tcm.2009.12.004.

3.     Rumyantsev P.P. Cardiomyocytes in reproduction, differentiation and regeneration. “Nauka”, 1982.

4.     H. Shih, BA, B. Lee, BA, R. J. Lee, MD, PhD, and A. J. Boyle, MBBS, PhD. The Aging Heart and Post-Infarction Left Ventricular Remodeling. J Am Coll Cardiol. 2010 December 28; 57(1): 9–17. doi:10.1016/j.jacc.2010.08.623.

5.     Shornikova M.V. Intermitochondrial contacts in cardiomyocyte mitochondriom in norm, under physiological overload and in pathology. Onthogenesis. – 2000. - ¹ 6. – Pp. 470-475

6.     J. N. Skepper and V. Navaratnam. Lipofuscin formation in the myocardium of juvenile golden hamsters: an ultrastructural study including staining for acid phosphatase. J. Anat. (1987), 150, pp. 1554167

7.     David B. Slautterback, Ph.D. Mitochondria in cardiac muscle cells of the canary and some other birds. The Journal of Cell Biology, Volume 4, 1965.

8.     Strehler BL, Mark DD, Mildvan AS. GEE MV: Rate and magnitude of age pigment accumulation in the human myocardium. J Gerontol. 1959 Oct; 14:430-9.