Insulin responsiveness of CK-M and CK-B mRNA in the diabetic rat heart.

Decreased cardiac performance is a known complication of diabetes mellitus, but the detailed molecular mechanisms that are responsible for this contractile abnormality are only incompletely explored, and cardiac gene products of known function, which are markedly and actively insulin responsive, have not been described. Recently, we found that creatine kinase (CK) enzyme activity and CK-M subunit mRNA levels are decreased in the heart of rats with experimental diabetes mellitus. These abnormalities could be restored to normal with chronic insulin administration. The CK-M and CK-B genes are expressed in the heart, and we wanted to determine whether diabetes also induces a change in CK-B mRNA levels. Quantitation of CK-M and CK-B mRNA levels on Northern blots with specific cDNA probes showed that, in diabetic hearts, CK-B mRNA levels represent only 19.8% of control levels and are more markedly depressed than CK-M mRNA levels, which are 46.5% of control values. Acute injection of insulin led to a significant 1.6-fold increase in CK-M mRNA and a 2.2-fold increase of CK-B mRNA 5 h after insulin injection. CK-M mRNA levels were restored to normal within 12 h, but 48 h were required to restore CK-B mRNA levels to normal values. After 1 mo of insulin therapy, CK-B mRNA levels had risen 9.7-fold, exceeding normal values by 90%, whereas CK-M mRNA levels were at the normal level as previously shown. CK enzyme activity showed only a small response to insulin administration 48 h postinjection. Diabetes leads therefore to a marked lowering of CK-M and CK-B mRNA levels in the rat heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-induced decreases in the messenger RNA coding for the sarcoplasmic reticulum Ca2(+)-ATPase of the rat heart.

Age-associated slowing of cardiac relaxation related to the decline in the Ca2+ pump function of cardiac sarcoplasmic reticulum (SR) has been previously described. It is unclear if the decreased Ca2+ pump function results from a lower amount of Ca2(+)-ATPase protein or a decreased pumping activity of the enzyme. To determine if these alterations could be mediated by changes in the amount of the protein itself, the level of the messenger RNA (mRNA) coding for the Ca2(+)-ATPase of the SR of Fischer rat hearts (4- and 30-month-old rats) were quantitated with a Northern blotting technique. We observed that the levels of SR Ca2(+)-ATPase mRNA were 60% lower in old rats as compared with young rats, suggesting that a quantitative reduction in the levels of the corresponding protein could occur during aging to explain the delayed diastolic relaxation documented in old animals as opposed to a change in the specific activity of this enzyme. The thyroid hormone responsiveness of SR Ca2(+)-ATPase mRNA has been previously established. We have found in this study that the thyroxine levels were consistently lower in old rats; however, this difference was relatively small (4.3 +/- 0.7 and 3.1 +/- 0.8 micrograms/dl [mean +/- SD), respectively, in young and old rats). In addition, no age-induced decrease in 3,5,3'-triiodothyronine levels was observed, suggesting that the aging process itself may be responsible for the changes in SR Ca2(+)-ATPase mRNA levels.

Diabetes decreases creatine kinase enzyme activity and mRNA level in the rat heart.

Several of the adenosinetriphosphatase enzymes that are responsible for cardiac muscle contraction rely on high-energy phosphates supplied by the creatine kinase (CK) system. Experimental diabetes mellitus has been shown to cause a decrease in the maximal contractile performance of the heart. We postulated that the decrease in contractile performance may be explained in part by a decrease in CK enzyme activity. To evaluate this possibility, we determined the level of CK activity and isoenzyme distribution in ventricular homogenates from normal, diabetic, and insulin-treated diabetic rats. We found that total CK activity was decreased by 35% in diabetic hearts and that a 66% reduction in the cardiac-specific MB isoenzyme occurs. Using a cDNA probe for CK-muscle (M) RNA in Northern blot analysis, we determined that a 61.1% decrease in CK-M mRNA occurs in diabetes. Chronic insulin therapy for 1 mo restores CK-M mRNA levels and enzyme activity. In conclusion, diabetes-induced CK enzyme decreases are mediated in part by a lower level of CK-M mRNA that codes for the major CK-M subunit protein. Decreased performance of the CK system may contribute to diabetic cardiomyopathy.

Ischemia induces changes in the level of mRNAs coding for stress protein 71 and creatine kinase M.

Hyperthermia, hypoxia, and other conditions induce the appearance of heat shock or stress proteins in cells. We have previously shown that in the ischemic dog myocardium the level of a messenger RNA (mRNA) coding for a protein with migration characteristics similar to heat shock/stress protein 71 increases. Using a human heat-shock protein (hHSP) 70 genomic clone and anti-HSP70 antibodies as probes, we demonstrate in this report that heart stress protein (SP) 71 mRNA and its translational products (71 kDa polypeptides) are members of the stress protein family. In rabbit hearts, the ischemia-induced mRNAs translate into three isoforms with different isoelectric points (6.0, 6.1, and 6.15), in contrast to dog heart mRNA that translates into a protein with a pI of 5.8. The levels of SP71 mRNA in the dog and rabbit ischemic myocardium increased by sixfold and 18-fold, respectively. In the same samples, the levels of creatine kinase M mRNA decreased by about 40%, whereas those of myosin heavy chain mRNA remain unaltered. Our comparative analysis of three different mRNAs indicates that ischemia manifests its effects by differentially changing the levels of specific mRNAs coding for proteins with separate and distinct roles in the cell.