Randa Hilal-Dandan

Small Heat Shock Proteins and Protection Against Ischemic Injury in Cardiac Myocytes

BACKGROUND Overexpression of the inducible hsp70 protects against ischemic cardiac damage. However, it is unclear whether the small heat shock proteins hsp27 and alphaB-crystallin protect against ischemic injury. METHODS AND RESULTS Our aim was to examine whether the overexpression of hsp27 and alphaB-crystallin in neonatal and adult rat cardiomyocytes would protect against ischemic injury. Recombinant adenovirus expressing hsp27 or alphaB-crystallin under the control of the cytomegalovirus promoter was used to infect cardiac myocytes at high efficiency as assessed by immunostaining. Overexpression was confirmed by Western blot analysis. Cardiomyocytes were subjected to simulated ischemic stress, and survival was estimated through assessment of lactate dehydrogenase and creatine phosphokinase release. The hsp27 overexpression decreased lactate dehydrogenase release by 45+/-7.5% in adult cardiomyocytes but had no effect in the neonatal cells. In contrast, alphaB-crystallin overexpression was associated with a decrease in cytosolic enzyme release in both adult (29+/-6.6%) and neonatal (32+/-5.4%) cardiomyocytes. Decreased endogenous hsp25 with an antisense adenovirus produced a 29+/-9.9% increase in damage with simulated ischemia. Overexpression of the inducible hsp70 in adult cardiomyocytes was associated with a 34+/-4.6% decrease in lactate dehydrogenase release and is in line with our previous results in neonatal cardiomyocytes. CONCLUSIONS The increased expression of hsp27 and alphaB-crystallin through an adenovirus vector system protects against ischemic injury in adult cardiomyocytes. Likewise, the overexpression of alphaB-crystallin protects against ischemic damage in neonatal cardiomyocytes. Decreasing the high levels of endogenous hsp25 present in neonatal cardiomyocytes renders them more susceptible to damage caused by simulated ischemia.

Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation.

The Ca2+ ATPase of the sarcoplasmic reticulum (SERCA2) plays a dominant role in lowering cytoplasmic calcium levels during cardiac relaxation and reduction of its activity has been linked to delayed diastolic relaxation in hypothyroid and failing hearts. To determine the contractile alterations resulting from increased SERCA2 expression, we generated transgenic mice overexpressing a rat SERCA2 transgene. Characterization of a heterozygous transgenic mouse line (CJ5) showed that the amount of SERCA2 mRNA and protein increased 2. 6-fold and 1.2-fold, respectively, relative to control mice. Determination of the relative synthesis rate of SERCA2 protein showed an 82% increase. The mRNA levels of some of the other genes involved in calcium handling, such as the ryanodine receptor and calsequestrin, remained unchanged, but the mRNA levels of phospholamban and Na+/Ca2+ exchanger increased 1.4-fold and 1.8-fold, respectively. The increase in phospholamban or Na+/Ca2+ exchanger mRNAs did not, however, result in changes in protein levels. Functional analysis of calcium handling and contractile parameters in isolated cardiac myocytes indicated that the intracellular calcium decline (t1/2) and myocyte relengthening (t1/2) were accelerated by 23 and 22%, respectively. In addition, the rate of myocyte shortening was also significantly faster. In isolated papillary muscle from SERCA2 transgenic mice, the time to half maximum postrest potentiation was significantly shorter than in negative littermates. Furthermore, cardiac function measured in vivo, demonstrated significantly accelerated contraction and relaxation in SERCA2 transgenic mice that were further augmented in both groups with isoproterenol administration. Similar results were obtained for the contractile performance of myocytes isolated from a separate line (CJ2) of homozygous SERCA2 transgenic mice. Our findings suggest, for the first time, that increased SERCA2 expression is feasible in vivo and results in enhanced calcium transients, myocardial contractility, and relaxation that may have further therapeutic implications.

Effects of Mutant and Antisense RNA of Phospholamban on SR Ca2+-ATPase Activity and Cardiac Myocyte Contractility

BACKGROUND The delayed cardiac relaxation in failing hearts has been attributed to a reduced activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2). Phospholamban (PLB) inhibits SERCA2 activity and is therefore a potential target to improve the cardiac performance in heart failure. METHODS AND RESULTS Mutants of PLB (Adv/mPLB) or antisense RNA of PLB (Adv/asPLB) was expressed in cardiac myocytes by recombinant adenovirus, and their effects on SERCA2 activity and myocyte contractility were studied. One mPLB, K3E/R14E, pentamerized with endogenous PLB in neonatal myocytes and resulted in a 45% increase in the affinity of SERCA2 for Ca(2+) and 27% faster diastolic Ca(2+) decline as determined by SR (45)Ca uptake assays and by indo 1-facilitated Ca(2+) transient measurement, respectively. Edge-detection analysis of adult myocyte contractility showed a 74% increase in fractional shortening, accompanied by 115% increase in velocity of relengthening and 25% decrease in time to half-maximal relengthening. In parallel, infection of neonatal cardiac myocytes by Adv/asPLB decreased the endogenous PLB level by 54%, which was associated with a 35% increase in Ca(2+) affinity of SERCA2 and 21% faster diastolic Ca(2+) decline. However, in adult cardiac myocytes, Adv/asPLB failed to significantly alter the endogenous PLB level, the SERCA2 activity, or most of the contractile parameters. CONCLUSIONS K3E/R14E is a dominant negative mutant of PLB that disrupts the structural integrity and function of the endogenous PLB and consequently enhances SERCA2 activity and myocyte contractility. In neonatal myocytes, the decrease in steady-state abundance of PLB by asPLB also leads to increased SERCA2 activity.

α1-Adrenergic Stimulation Inhibits 3,5,3′-Triiodothyronine-Induced Expression of the Rat Heart Sarcoplasmic Reticulum Ca2+ Adenosine Triphosphatase Gene1

The interactions between the β-adrenergic system and thyroid hormone (T3) on cardiac function have been investigated in detail. In addition to β-adrenoceptors, α1-adrenergic receptors are present in the mammalian heart. The interactions between T3 and the α1-adrenergic system remain, however, poorly understood. T3 stimulates the expression and transcription of the sarcoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA2) gene, a protein vital in the control of cardiac calcium transients and contractility. We show that in rat cardiac myocytes, the stimulatory effect of T3 on SERCA2 messenger RNA expression and gene transcription is inhibited by anα 1-adrenergic agonist. We demonstrate that direct activation of the α1-adrenergic signaling pathway, using a mutant constitutively active G protein (Gq) similarly down-regulated the T3 effect on SERCA2 transcription. The combined effect of thyroid hormone receptor and retinoid X receptors on T3-stimulated SERCA2 gene transcription was also markedly attenuated...

Endothelin downregulates SERCA2 gene and protein expression in adult rat ventricular myocytes : regulation by pertussis toxin-sensitive Gi protein and cAMP

Downregulation of the sarcoplasmic reticulum calcium ATPase (SERCA2) is associated with diastolic dysfunction in the failing heart. Elevated plasma endothelin-1 (ET) levels are correlated with congestive heart failure suggesting that ET may play a pathophysiological role. We have investigated the ability of ET to regulate SERCA2 gene expression in isolated adult rat ventricular myocytes. We find that ET enhances net protein synthesis by approximately 40% but significantly downregulates SERCA2 mRNA expression, time dependently, by approximately 30-50%, and the expression of SERCA2 protein by approximately 50%. In myoyctes, ET binds to ET(A) receptor that couples to G(q) and G(i) proteins. Inhibition of G(q)-PLC-induced phosphoinositide (PI) hydrolysis with U73122 (1 muM) or inhibition of G(i) protein with pertussis toxin (PTX) abolishes the ability of ET to downregulate SERCA2 mRNA gene expression. Further investigation suggests that ET coupling to PTX-sensitive G(i) with consequent lowering of cAMP is required for downregulation of SERCA2 mRNA levels. Increasing intracellular cAMP quantity using cAMP-specific PDE inhibitor Ro20-1724 or cAMP analog dibutyryl-cAMP reverses ET-induced downregulation of SERCA2 mRNA levels. The data indicate that, in adult myocytes, ET downregulates SERCA2 mRNA and protein levels, and the effect requires cross-talk between G(q) and PTX-sensitive G(i) pathways.