Phospholamban mRNA Expression Research Articles

Abstract 530: Mechanisms Underlying Phospholamban L39 Stop (PLN L39X) Cardiomyopathy

Background: Defective calcium (Ca++) handling is a hallmark of HF across species. Together with the Sarco/Endoplasmic Reticulum Ca-ATPase (SERCA2a), Phospholamban (PLN) has emerged as a critical regulator of Ca++homeostasis. Worldwide, PLN mutations are identified with increasing frequency in patients with dilated, hypertrophic and arrhythmogenic cardiomyopathy (CMPs) but the causative defects leading to the CMP remain incompletely understood. While preclinical studies have unequivocally shown that absence of PLN (PLNKO) is therapeutic in rodent HF models, the discovery of a human pathogenic mutation (L39X) presumed to be the human equivalent of the PLNKO lead to the conclusion that PLN ablation was lethal in human, mitigating any enthusiasm in targeting PLN inhibition in PLN-associated disease or HF treatment. The objective of this proposal is to levarge the use of “induced-pluripotent” stem cells (iPSCs) derived cardiomyocytes (CM) from homozygote L39X carriers to elucidate the role of PLN (L39) in human pathophysiology. Methodology and Results: We obtained mononuclear cells from Homozygotes (Hom) L39 carriers and generated 11 iPSC clones. To derive CMs, we used the direct differentiation method temporally modulating the Wnt/β-catenin signaling. Immunocytostaining revealed positive expression of cardiac troponin T as well as PLN. In Hom L39 derived CMs, PLN showed an abnormal cytoplasmic distribution, formed intracellular aggregates and there was loss of perinuclear localization when compared to matched WT iPSC-CMs. Using fura-2AM, we observed decreased calcium transient amplitude in iPSC-CMs from L39 compared to WT with prolongation of the time constant of relaxation and early after depolarization (EAD). Lastly, we saw a 70% and 50% reduction in the protein and mRNA expression of PLN and SERCA2a respectively. Conclusions: Our data suggest that the L39 PLN mutant is expressed but mis-located within the cardiomyocytes. The mis-location of PLN was associated to decreased SERCA2a expression impaired Ca++ handling and increased arrhythmogenicity. Further studies will be required to fully elucidate the impact of the mutation in HF pathophysiology

How Does Glucose Insulin Potassium Improve Hemodynamic Performance?

Glucose insulin potassium (GIK) improves hemodynamic performance after coronary artery surgery (CABG). We investigated whether this is associated with changes in gene expression of beta1-adrenergic receptor (ADRB1) or other calcium handling proteins. During a randomized double-blind placebo-controlled trial, 48 patients undergoing on-pump CABG, allocated to receive pre-ischemic placebo (5% dextrose) or GIK (40% dextrose, K+ 100 mmol.L(-1), insulin 70 u.L(-1); 0.75 mL.kg(-1).h(-1)) continued for 6 hours after the removal of the aortic cross-clamp (AXC), underwent left ventricular biopsy for analysis of specific mRNAs immediately before AXC, before release of AXC, and 10 minutes after reperfusion (placebo n=24, GIK n=24). GIK or placebo was infused for a mean of 79+/-21 minutes or 79+/-18 minutes pre-ischemia respectively. Serial hemodynamic measurements were performed. Biopsy samples were snap-frozen and stored at -80 degrees C, mRNA was extracted and TaqMan real-time polymerase chain reaction was performed to investigate expression of ADRB1, sarcoplasmic reticulum Ca-ATPase (SERCA2a), and phospholamban (PLB). GIK significantly increased cardiac index versus placebo (P=0.037). TaqMan reverse-transcriptase polymerase chain reaction showed significantly greater ADRB1 mRNA expression at all time points (4.9-fold, 7.4-fold, and 15.6-fold increase, respectively; P<0.001), significantly greater SERCA2a mRNA expression after reperfusion (13.2-fold; P<0.001), and increased PLB mRNA expression at pre-ischemia and reperfusion (P<0.001 for both time-points) in GIK groups versus placebo. The beneficial hemodynamic effects of GIK therapy are associated with increased ADRB1 and SERCA2a mRNA expression. Further work is therefore warranted to investigate these mRNA effects at the protein level.

Decreased cardiac sarcoplasmic reticulum Ca2+-ATPase activity contributes to cardiac dysfunction in streptozotocin-induced diabetic rats

Diabetic cardiomyopathy is characterized by reduced cardiac contractility due to direct changes in myocardium function independent of vascular disease. This study is to investigate the alterations of cardiac sarcoplasmic reticulum Ca2+ -ATPase activity and cardiac function in streptozotocin-induced diabetic rats. Diabetes mellitus (DM) was induced in male Wistar rats by intraperitoneal injection of streptozotocin. The activity of myocardium sarcoplasmic reticulum Ca2+ -ATPase and the left ventricular hemodynamic parameters were measured in DM rats 4 weeks, 6 weeks and 8 weeks after streptozotocin was administered. Phospholamban mRNA expression was detected by reverse transcription-polymerase chain reaction, and the protein levels of phospholamban and sarcoplasmic reticulum Ca2+ -ATPase were determined by Western blot. Normal rats served as control group. It was found that in DM rats 4 weeks after streptozotocin injection, the cardiac function, myocardium sarcoplasmic reticulum Ca2+ -ATPase activity, phospholamban mRNA and phospholamban protein were not significantly changed compared with those in the control rats. At 6 and 8 weeks after the streptozotocin injection, DM rats showed a significant decrease in sarcoplasmic reticulum Ca2+ -ATPase activity and cardiac function, as indicated by an increase of LVEDP and a marked depression in LVSP and +/- dP/dtmax. At the same time points, increases in phospholamban mRNA and protein levels were observed in DM rats. Sarcoplasmic reticulum Ca2+ -ATPase protein level showed no significant alterations in all DM rats compared with that in control rats. Our work confirms that sarcoplasmic reticulum Ca2+ -ATPase activity is depressed in rats with streptozotocin-induced DM, which is accompanied by elevated phospholamban protein level thus contribute to the pathogenesis of cardiac dysfunction in diabetic rats.

Effects of simvastatin on cardiac performance and expression of sarco-plasmic reticular calcium regulatory proteins in rat heart

To investigate the effect of simvastatin on the cardiac contractile function and the alteration of gene and protein expression of the sarcoplasmic calcium regulatory proteins, including sarcoplasmic reticulum Ca2+-ATPase (SERCA), phospholamban (PLB), and ryanodine receptor 2 (RyR2) in rat hearts. Langendorff-perfused rat hearts were subjected to 60-min perfusion with different concentrations of simvastatin (1, 3, 10, 30, or 100 microml/L), and the parameters of cardiac function such as left ventricular developed pressure (LVDP), +dp/dtmax, and -dp/dtmax were determined. The cultured neonatal rat ventricular cardiomyocytes were incubated with simvastatin (1, 3, 10, 30, and 100 micromol/L) for 1 h or 24 h. The levels of SERCA, PLB, and RyR2 expression were measured by reverse transcription-polymerase chain reaction and Western blot. Cytotoxic effect of simvastatin on ventricular cardiomyocytes was assessed by the MTT colorimetric assay. LVDP, +dp/dtmax, and -dp/dtmax of hearts were increased significantly after treatment with simvastatin 3, 10, and 30 micromol/L. In simvastatin-treated isolated hearts, the levels of mRNA expression of SERCA and RyR2 were elevated compared with the control (P<0.05), while the mRNA expression of PLB did not change. After the cultured neonatal rat ventricular cardiomyocytes were incubated with 3, 10, 30, and 100 mumol/L simvastatin for 1 h, SERCA and RyR2 mRNA expressions of cardiomyocytes rose, but there was no alteration in protein expressions. However, with the elongation of simvastatin treatment to 24 h, the protein expression of SERCA and RyR2 were also elevated. Additionally, simvastatin (1-30 micromol/L) had no influence on cell viability of cultured cardiac myocytes, but simvastatin 100 micromol/L inhibited the cell viability. Simvastatin improved cardiac performance accompanied by the elevation of SERCA and RyR2 gene and protein expression.

Suppressed enhancement of the force-frequency relation to adrenergic stimulation is an indicator of poor prognosis in patients with dilated cardiomyopathy

Objectives: To investigate the relation between the response of force-frequency relation (FFR) to adrenergic stimulation, and gene expression of Ca 2+-handlin proteins in patients with dilated cardiomyopathy (DCM). Methods and Results: We measured the maximum first derivative of left ventricular (LV) pressure (LV dP/dt max) during pacing and dobutamine infusion in 10 patients with mild DCM (NYHA I-II). Endomyocardial biopsy was performed, and mRNA levels of SR Ca 2+-ATPase(SERCA2a), ryanodine receptor-2, phospholamban (PLB), calsequestrin, and Na +/ Ca2 + exchanger were quantified by a real-time quantitative reverse transcription-polymerase chain reaction method. In 6 patients (group A), the increase in LV dP/dt max during dobutamine infusion was greater than pacing alone, whereas in 4 patients (group B), the increase in LV dP/dt max during dobutamine infusion was significantly attenuated compaired with group A. The ratio of SERCA2a to PLB mRNA expression was significantly higher in group B than group A. In group B, one patient has died, and two has hospitalized for congestive heart failure during follow-up period (25 ± 18months). Conversely, there were no death and hospitalization in group A. Conclusions: In this study, the LV contractile reserve to adrenergic stimulation was impaired in some patients with mild DCM, and those showed poor prognosis, although the ratio of SERCA2a to PLB mRNA expression was in normal range.

Phospholamban ablation by RNA interference increases Ca 2+uptake into rat cardiac myocyte sarcoplasmic reticulum

Phospholamban (PLB) inhibits SR Ca 2+-ATPase 2 (SERCA2) Ca 2+ uptake and is a potential therapeutic target in the context of heart failure. RNA interference (RNAi) is a technique that produces sequence-specific, post-transcriptional gene silencing through the use of double-stranded RNA directed against the homologous target gene. The goal of the current study was to investigate the efficacy of the RNAi method for ablation of PLB gene expression and restoration of Ca 2+ uptake function in cultured neonatal rat cardiac myocytes in which SERCA2 protein levels were decreased. Myocytes were transfected with 21-nucleotide duplexes of small interfering RNA (siRNA) targeting PLB (30 nmol/l) or with scramble sequence using a haemagglutinating virus of Japan (HVJ) envelope vector. Administration of PLB siRNA resulted in the reduction of PLB mRNA level to approximately 6% of that observed after administration of scramble siRNA group at 12 h after transfection. Further, PLB protein levels in the PLB siRNA groups were 12% of that in cells treated with scramble siRNA on day 2, and the mRNA and protein levels for SERCA2 and calsequestrin were not affected. In addition, Ca 2+ uptake affinity was increased in total homogenates from the PLB siRNA group (a 29% decrease in EC 50 value when compared with scramble siRNA group). Finally, PLB siRNA restored Ca 2+ uptake affinity following hydrogen peroxide-induced decreases in SERCA2 and PLB mRNA expression. These results demonstrate that PLB-targeted RNAi inhibited endogenous PLB expression in neonatal rat myocytes and restored Ca 2+ uptake affinity in cardiac myocytes in which SERCA2 protein levels were decreased. This technique may represent a novel therapeutic strategy for heart failure.

In vitro and in vivo promoter analyses of the mouse phospholamban gene

To determine the mechanisms responsible for regulation of the phospholamban ( PLB) gene expression, a critical regulatory phosphoprotein in cardiac muscle, the mouse PLB gene was isolated and promoter analysis was performed in vitro and in vivo. The PLB gene consists of two exons separated by a single large intron. Deletion analysis revealed that a 7-kb 5′ flanking fragment (including exon 1, the entire intron and part of exon 2) was necessary for maximal transcriptional activity in H9c2 and L6 cell lines. Interestingly, deletion of a 2.4-kb intronic region, which contained repetitive elements, caused a dramatic increase in CAT activity in both these cell lines. In vivo analysis indicated that the PLB fusion gene containing 7 kb of the 5′-flanking region was capable of cardiac specific gene expression in transgenic mice. Furthermore, these mice exhibited 3-fold higher levels of CAT activity in the ventricles compared with the atria, mimicking endogenous PLB mRNA expression. Our findings suggest that: (a) PLB gene expression may be regulated by the interplay of cis-acting regulatory elements located within the 5′ flanking and intronic regions; and (b) the 7-kb upstream region is capable of directing cardiac-specific and compartment-specific expression in vivo.

Regulation of myocardial Ca2+-ATPase and phospholamban mRNA expression in response to pressure overload and thyroid hormone.

The sarcoplasmic reticulum (SR) and the contractile protein myosin play an important role in myocardial performance. Both of these systems exhibit plasticity--i.e., quantitative and/or qualitative reorganization during development and in response to stress. Recent studies indicate that SR Ca2+ uptake function is altered in adaptive cardiac hypertrophy and failure. The molecular basis (genetic and phenotypic) for these changes is not understood. In an effort to determine the underlying causes of these changes, we characterized the rabbit cardiac Ca2+-ATPase phenotype by molecular cloning and ribonuclease A mapping analysis. Our results show that the heart muscle expresses only the slow-twitch SR Ca2+-ATPase isoform. Second, we quantitated the steady-state mRNA levels of two major SR Ca2+ regulatory proteins, the Ca2+-ATPase and phospholamban, to see whether changes in mRNA content might provide insight into the basis for functional modification in the SR of hypertrophied hearts. In response to pressure overload hypertrophy, the relative level of the slow-twitch/cardiac SR Ca2+-ATPase mRNA was decreased to 34% of control at 1 week. The relative Ca2+-ATPase mRNA level increased to 167% of control after 3 days of treatment with thyroid hormone. In contrast, in hypothyroid animals, the relative Ca2+-ATPase mRNA level decreased to 51% of control at 2 weeks. The relative level of phospholamban mRNA was decreased to 36% in 1-week pressure overload. Hyperthyroidism induced a decrease to 61% in the phospholamban mRNA level after 3 days of treatment, while hypothyroidism had virtually no effect on phospholamban mRNA levels. These data indicate that the expression of SR Ca2+-ATPase and phospholamban mRNA may not be coordinately regulated during myocardial adaptation to different physiological conditions.