EXPRESSION OF MARKERS CD34 AND CD77 ON THE PLASMA MEMBRANE OF HYPERCALCEMIC RATS HEART CELLS

UVOD: Depolarizacijski signal koji je započet na površini mišićnog vlakna, potiče oslobađanje Ca2+ iz cisterne endoplazmatske mrežice i tako se širi kroz cijelo mišićno vlakno. Nakon depolarizacije, membrane sarkoplazmatske mrežice živčanim podražajem, ioni Ca2+ koji su nakupljeni u cisternama sarkoplazmatske mrežice, pasivno se ispuštaju u blizini područja prekrivanja debelih i tankih filamenata, te se tu vežu za troponin i omogućuju povezivanje aktina s miozinom. Kada se depolarizacija membrane završi, sarkoplazmatska mrežica odvodi kalcij koji se aktivno vraća u cisterne pa kontrakcija prestaje. Poznato je da pri hiperkalcijemiji u srčanom mišiću, dolazi do skraćivanja refraktornog razdoblja i do usporenog provođenja srčanih impulsa što rezultira pojavama aritmija. Uz aritmije, hiperkalcijemija potiče kontrakciju srca i uzrokuje hipertenziju. CD34 stanice koriste se kao biljezi za identifikaciju i izolaciju nekih hematopoetskih matičnih stanica i prastanica. Kaveole bubrežnih endotelnih i epitelnih plazma membrana posebice obiluju u sadržaju neutralnog glikosfingolipida CD77. CILJ ISTRAŽIVANJA: Odrediti izražaj CD34 i CD77 biljega na plazma membrani srčanih stanica hiperkalcijemičnih štakora u usporedbi s izražajem na plazma membrani srčanih stanica kontrolnih štakora. MATERIJALI I METODE: Mužjaci štakorskog soja Sprague-Dawley, starosti 6 tjedna bili su podijeljeni u 2 skupine: ispitivanu skupinu koja je podvrgnuta hiperkalcijemičnom tretmanu (n=8) i netretiranu kontrolnu skupinu (n=9). Da bi postigli hiperkalcijemiju, hiperkalcijemičnoj skupini se davala voda koja je bogata CaCl2. Period u kojem su štakori pili vodu obogaćenu CaCl2 bio je 14 dana. Štakorima iz kontrolne i hiperkalcijemične skupine, nakon žrtvovanja, izvađena su tkiva srca. Sve životinje žrtvovane su produljenom anestezijom (dietil eter). Nakon toga pripremljene izolirane stanice srca inkubirane su sa dva protutijela za dvostruko bojenje stanica: protutijelo na CD34 konjugirano s PC5 i CD77 konjugirano s FITC-om, te su analizirane protočnim citometrom. REZULTATI: Postotak ioniziranog kalcija u hiperkalcijemičnoj skupini iznosio je 48 ± 0.30 % a u kontrolnoj skupini 44 ± 0.96 % (P=0.013). U srcu hiperkalcijemičnih štakora udio CD34+CD77- stanica je blago snižena u odnosu na kontrolnu skupinu. ZAKLJUČAK: Sniženje postotka CD34+CD77- srčanih stanica kod hiperkacijemičnih štakora u odnosu na kontrolnu skupinu nije bilo statistički značajno, ali ovi podatci potvrđuju najnovija istraživanja koja su pokazala da je cijepanje vaskularnog endotelnog kadherina uzrokovano priljevom Ca2+.INTRODUCTION: The depolarisation signal is initiated on the surface of the muscle fibers, then it stimulates the release of Ca2+ from the endoplasmic reticulum cisterns and thus spreads through the muscle fiber. After the depolarization, the membrane of the sarcoplasmic meshes stimulated with the nerve stimulation, the Ca2+ ions that have accumulated in tanks of sarcoplasmic mesh, discharge passive into the nearby areas of the overlapping thick and thin filaments, and there they are bound to troponin and allow actin to connect with myosin. When the depolarization of the membrane is complete, the sarcoplasmic mesh drains calcium that actively returns to the tank and than the contraction is ceased. It is known that hypercalcemia in the cardiac muscle leads to shortening of the refractory period and to the slow implementation of the cardiac impulse which results in the occurrence of arrhythmias. With arrhythmia, hypercalcemia also stimulates the contractions of the heart and can cause hypertension. CD34 cells are used as markers for the identification and isolation of certain hematopoietic stem and progenitor cells. Caveolae of renal endothelial and epithelial plasma membranes are particularly abundant with neutral glycosphingolipid CD77. AIM OF RESEARCH: To determine the expression of CD34 and CD77 markers on the plasma membrane of cardiac cells in hypercalcemic rats compared with the expression on the plasma membrane of cardiac cells in control rats. MATERIALS AND METHODS: Male Sprague – Dawley rats, 6 weeks old separated in 2 groups: the examination group undergone the hypercalcemic treatment (n=8) and untreated controlled group (n=9). To achieve hypercalcaemia, to the hypercalcemic group was given water that was rich with CaCl2. The period in which the rats were drinking water enriched with CaCl2 was 14 days. After sacrifice, the heart tissue was removed from the rats in the control group and in the hypercalcemic group. All animals were sacrificed in prolonged anesthesia (diethylether). The subsequently prepared isolated heart cells were incubated with two antibodies for the double staining of cells: CD34 antibody conjugated with PC5 and CD77 conjugated with FITC, then analyzed by flow cytometry. RESULTS: Percentage of ionized calcium in the hypercalcemic group was 48 ± 0.30% and in the control group 44 ± 0.96% (P = 0.013). In the heart of hypercalcemic rats the share of CD34 + CD77- cells were slightly reduced compared to the control group. CONCLUSION: Reduction in the percentage of CD34+CD77- cells in the heart of hypercalcemic rats with regard to the control group was not statistically significant, but these data confirm the latest researches which have shown that the splitting of vascular endothelial cadherin was caused by the influx of Ca2+