EFEK PEMBERIAN KALSITRIOL (satu alfa dua lima dihidrivitamin d tiga) TERHADAP KEMAMPUAN KONTRAKSI KULTUR KARDIOMIOSIT Kajian terhadap fosforilasi troponin I dan kadar cyclic AMP

CUT SIDRAH NADIRA, Dr. dr. Denny Agustiningsih, M.Kes, AIFM; Prof. dr. Sri Kadarsih S, M.Sc,Ph.D, AIFM

2015 | Tesis | S2 Ilmu Kedokteran Dasar dan Biomedis

Latar belakang: Defisiensi vitamin D meningkatkan faktor risiko terjadinya penyakit kardiovaskular. Kalsitriol (bentuk aktif vitamin D) melalui fosforilasi troponin I berperan dalam regulasi respon frekuensi dan kekuatan kontraksi pada proses ejeksi ventrikel. Jalur mekanisme fosforilasi troponin I (TnI) salah satunya melalui cAMP/PKA. Penelitian ini bertujuan untuk mempelajari efek kalsitriol terhadap peningkatan fosforilasi TnI serta jalur mekanisme aksinya yang melalui cAMP/PKA. Metode: Penelitian ini merupakan penelitian eksperimental murni menggunakan rancangan pre-post test with control group dengan sampel kultur primer kardiomiosit Sprague-Dawley jantan dewasa yang dibagi dalam 6 kelompok. Kardiomiosit diinkubasi kalsitriol selama 5 menit kemudian fosforilasi TnI dinilai dengan teknik immunocytochemistry (ICC) menggunakan antibodi phospho-Ser22+Ser23 dan jumlah sel dengan TnI yang terfosforilasi dihitung menggunakan software image raster. Kadar cAMP diukur menggunakan metode ELISA. Hasil Penelitian: Perbedaan fosforilasi TnI pre dan post test yang bermakna (p<0,05) dijumpai pada setiap kelompok dengan nilai rerata ± SD (intensitas kuat, sedang dan lemah secara berurutan) sebagai berikut: pretest: (7,83 ± 4,08)%; (26,27 ± 3,00)%; (65,89 ± 2,46)%, K(-): (2,89 ± 2,65)%; (25,9 ± 8,48)%; (71,22 ± 10,67)%, D1: (21,57 ± 13,86)%; (54,75 ± 19,09)%; (23,69 ± 9,39)%, D2: (23,42 ± 10,33)%; (72,45 ± 10,49)%; (4,14 ± 3,35)%, D3: (29,92 ± 9,51)%; (61,09 ± 7,35)%; (8,99 ± 4,13)%, K(+): (57,05 ± 23,01)%; (34,95 ± 16,78)%; (8,01 ± 7,95)%. Kadar cAMP pre dan post test setiap kelompok tidak mengalami peningkatan, dengan nilai rerata ± SD sebagai berikut: pretest (307,67 ± 47,78 pmol/mL); K(-) (285,92 ± 46,63 pmol/mL); D1 (282,86 ± 19,41 pmol/mL); D2 (209,39 ± 33,58 pmol/mL), D3 (188,93 ± 34,16 pmol/mL) dan K(+) (175,20 ± 6,64 pmol/mL). Tidak dijumpai korelasi yang bermakna antara peningkatan fosforilasi TnI dengan penurunan kadar cAMP (p>0,05). Kesimpulan: Inkubasi kalsitriol selama 5 menit dapat meningkatkan fosforilasi TnI, akan tetapi jalur aktivasi cAMP/PKA oleh kalsitriol belum terbukti.

Background: Vitamin D deficiency is associated with increasing risk of cardiovascular diseases. Calcitriol (active form of vitamin D), through phosphorilated-cardiac troponin I (cTnI), contributes in regulation of ventricular ejection force and frequency response. One of calcitriol action mechanism in troponin I phosphorylation is through cAMP/PKA pathway. The aim of this research is to know the calcitriol effect on increasing cardiomyocytes phosphorylated-troponin I and the involvement of cAMP/PKA signaling cascade. Methods: This is a true experimental research with pre-post test with control group design. It used primary cultured cardiomyocytes of adult male Sprague-Dawley ventricles which was divided in 6 groups, as the samples. Cardiomyocytes was incubated in calcitriol for 5 minutes, then phosphorylated-troponin I was assessed with immunocytochemistry technique using phospho-Ser22+Ser23 antibody and then counted by image raster software. cAMP level was measured by ELISA method. Results: Significant difference of pre-post test phosphorylated-troponin I was observed in each groups (p<0,05). Mean ± SD of cardiomyocytes phosphorylated-troponin I as high, intermediate and low intensity for each groups consecutively shows below: pretest: (7,83 ± 4,08)%; (26,27 ± 3,00)%; (65,89 ± 2,46)%, K(-): (2,89 ± 2,65)%; (25,9 ± 8,48)%; (71,22 ± 10,67)%, D1: (21,57 ± 13,86)%; (54,75 ± 19,09)%; (23,69 ± 9,39)%, D2: (23,42 ± 10,33)%; (72,45 ± 10,49)%; (4,14 ± 3,35)%, D3: (29,92 ± 9,51)%; (61,09 ± 7,35)%; (8,99 ± 4,13)%, K(+): (57,05 ± 23,01)%; (34,95 ± 16,78)%; (8,01 ± 7,95)%. There was no increased of cAMP level in each groups, with mean ± SD of each groups as shown below: pretest (307,67±47,78 pmol/mL); K(-) (285,92 ± 46,63 pmol/mL); D1 (282,86 ± 19,41 pmol/mL); D2 (209,39 ± 33,58 pmol/mL), D3 (188,93 ± 34,16 pmol/mL) dan K(+) (175,20 ± 6,64 pmol/mL). There was also no significant correlation observed between increased phosphorylated-troponin I and decreased cAMP level in this research (p>0,05). Conclusion: Incubating in calcitriol for 5 minutes elevated cardiomyocytes phosphorylated-troponin I, nevertheless the involvement of cAMP/PKA signaling cascade in this action still uncertain.

Kata Kunci : Kalsitriol, kontraktilitas, kardiomiosit, cAMP, Troponin I, Calcitriol, contractility, cardiomtocytes, cAMP, phosphorilated-troponin I