Activation of creatine kinase-B and phospholamban gene expression in transformed latissimus dorsi muscle: evaluation of mRNA by polymerase chain reaction.
Latissimus dorsi muscle (LDM) transformation following chronic stimulation is the critical requirement for its use in cardiac assist procedures. In order to identify one or two molecular markers that can be used to effectively monitor the LDM transformation, the modulation in the expression of creatine kinase (CK) and phospholamban (PLB) genes by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was examined. Continuous in situ stimulation of left LDM was performed in four dogs for a period of 10 weeks after a vascular delay period of 2 weeks following surgery. For RT-PCR, gene-specific radiolabeled primers and equal amounts of cDNA synthesized from total RNA extracted from the LDM biopsies obtained at 4, 7, and 10 weeks of stimulation were used. A 2.6-fold increase in creatine kinase (brain type) (CK-B) mRNA was observed at transformed LDM compared to the control (P = 0.004) following 10 weeks of stimulation. On the contrary, a 30% decline was observed in creatine kinase (muscle type) (CK-M) mRNA level. An increase up to eight-fold was also observed in PLB mRNA in stimulated LDM compared to the contralateral muscle (P = 0.002). The PLB mRNA level in transformed LDM reached plateau and became comparable to that of normal heart after 7 weeks of stimulation. However, a sustained increase in CK-B mRNA level was observed until 10 weeks of stimulation. The level of beta-actin mRNA used as control remained the same in both stimulated and control samples. Thus the increase in CK-B and PLB mRNA and downregulation of CK-M mRNA in transformed LDM, demonstrated here by RT-PCR, indicate a switch from anaerobic to aerobic potential of transformed LDM along with a change towards slow-twitch phenotype and provide valuable markers to monitor the effectiveness of muscle transformation in cardiomyoplasty.
Division of Cardiology, State University of New York, Health Science Center at Brooklyn 11203, USA.
M
M
Alam
M
M
Vaynblat
S K
SK
Goswami
M M
MM
Baig
G
G
Grijalva
M
M
Chiavarelli
Z
Z
Zisbrod
I J
IJ
Jacobowitz
W
W
Cheng
R A
RA
Stein
M A
MA
Siddiqui
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001706:Biopsy; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D003402:Creatine Kinase / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D004789:Enzyme Activation; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis; D013997:Time Factors; D000099211:Phospholamban
D000199:Actins; D000251:Adenosine Triphosphatases; D002135:Calcium-Binding Proteins; D003402:Creatine Kinase; D012333:RNA, Messenger; D000099211:Phospholamban