Anne-Marie Lompré

Function of the sarcoplasmic reticulum and expression of its Ca2(+)-ATPase gene in pressure overload-induced cardiac hypertrophy in the rat.

The reduction in Ca2+ concentration during diastole and relaxation occurs differently in normal hearts and in hypertrophied hearts secondary to pressure overload. We have studied some possible molecular mechanisms underlying these differences by examining the function of the sarcoplasmic reticulum and the expression of the gene encoding its Ca2(+)-ATPase in rat hearts with mild and severe compensatory hypertrophy induced by abdominal aortic constriction. Twelve sham-operated rats and 31 operated rats were studied 1 month after surgery. Eighteen animals exhibited mild hypertrophy (left ventricular wt/body wt less than 2.6) and 13 animals severe hypertrophy (left ventricular wt/body wt greater than 2.6). During hypertrophy we observed a decline in the function of the sarcoplasmic reticulum as assessed by the oxalate-stimulated Ca2+ uptake of homogenates of the left ventricle. Values decreased from 12.1 +/- 1.2 nmol Ca2+/mg protein/min in sham-operated rats to 9.1 +/- 1.5 and 6.7 +/- 1.1 in rats with mild and severe hypertrophy, respectively (p less than 0.001 and p less than 0.001, respectively, vs. shams). This decrease was accompanied by a parallel reduction in the number of functionally active CA2(+)-ATPase molecules, as determined by the level of Ca2(+)-dependent phosphorylated intermediate: 58.8 +/- 7.4 and 48.1 +/- 13.5 pmol P/mg protein in mild and severe hypertrophy, respectively, compared with 69.7 +/- 8.2 in shams (p less than 0.05 and p less than 0.01, respectively, vs. shams). Using S1 nuclease mapping, we observed that the Ca2(+)-ATPase messenger RNA (mRNA) from sham-operated and hypertrophied hearts was identical. Finally, the relative level of expression of the Ca2(+)-ATPase gene was studied by dot blot analysis at both the mRNA and protein levels using complementary DNA clones and a monoclonal antibody specific to the sarcoplasmic reticulum Ca2(+)-ATPase. In mild hypertrophy, the concentrations of Ca2(+)-ATPase mRNA and protein in the left ventricle were unchanged when compared with shams (mRNA, 93.8 +/- 10.6% vs. sham, NS; protein, 105.5 +/- 14% vs. sham, NS). in severe hypertrophy, the concentration of Ca2(+)-ATPase mRNA decreased to 68.7 +/- 12.9% and that of protein to 80.1 +/- 15.5% (p less than 0.001 and p less than 0.05, respectively), whereas the total amount of mRNA and enzyme per left ventricle was either unchanged or slightly increased. The slow velocity of relaxation of severely hypertrophied heart can be at least partially explained by the absence of an increase in the expression of the Ca2(+)-ATPase gene and by the relative diminution in the density of the Ca2+ pumps.(ABSTRACT TRUNCATED AT 400 WORDS)

Myosin isoenzyme changes in several models of rat cardiac hypertrophy.

We studied the effect of chronic mechanical overloading on the isoenzymic composition of rat cardiac myosin in several experimental models: aortic stenosis (AS), aortic incompetence (AI), aortocaval fistula (ACF), overload of the non-infarcted area after left coronary ligation (INF), and overload of the spontaneously hypertensive rats (SHR). Samples of the left and right ventricles were isolated from these hearts, and myosins were analyzed by electrophoresis in non-dissociating conditions. The myosin isoenzymes were called VI, V2, and V3 in order of decreasing mobility, according to the nomenclature of Hoh et al. Controls of the Wistar and Wistar Kyoto (WKY) strains were almost exclusively VI. A slow age-dependent shift toward V3 was observed in the left ventricles of adult Wistar rats, which at 30 weeks of age (body weight 600 g) contained approximately 15% of this form. In all models of cardiac hypertrophy, an isoenzymic redistribution was observed with a significant increase in V3. The level of V3 was statistically correlated with the degree of hypertrophy in the AS, (ns 11, r - 0.6, P < 0.05), the AI (n = 14, r - 0.88, P< 0.001), and the AS + AI (n a 14, r= 0.69, P < 0.01) but not in the ACF (TJ = 16, r = 0.46). The isoenzymic changes could account for the decreases in both myosin ATPase activity and cardiac contractility described previously in our laboratory and by others. They also demonstrate that changes in myosin isoenzymes represent a general response of the rat heart, to chronic mechanical overloading.

Species- and age-dependent changes in the relative amounts of cardiac myosin isoenzymes in mammals

In mice, rabbits, and pigs, two basic types of cardiac myosin isoenzymes were found by electrophoresis of native molecules: a fast-migrating form with high Ca(2+)-dependent ATPase activity and a slow-migrating form with low activity. According to the nomenclature of J. F. Y. Hoh, P. A. McGrath, and P. T. Hale (1978, J. Mol. Cell. Cardiol. 10, 1053-1076) these forms are called, respectively, V1 and V3. In all species, myosin was essentially V3 during fetal life, while V1 appeared around the time of birth. There were species differences in adults: mice remained V1, while rabbits and pigs returned to V3 after 3 weeks of age. Adult dog, beef, and human myosins were also composed of the V3 form only.

Alteration in temporal kinetics of Ca2+ signaling and control of growth and proliferation

Abstract Calcium is a ubiquitous second messenger controlling a broad range of cellular functions including growth and proliferation. Quiescent, hyperthrophic and proliferating cells have different types of calcium signal. In quiescent cells the calcium signal mostly involves elementary calcium events such as sparks and puffs, produced by localized Ca2+ release via a cluster of intracellular calcium channels, IP3 receptors and ryanodine receptors. This type of calcium signal promotes activation of the transcription factor CREB (cAMP response element binding protein) leading to cell cycle arrest in G1 phase via transactivation of p53/p21 signaling pathways. Proliferation is induced by phosphoinositide‐coupled agonists and is associated with a sustained increase in cytosolic calcium due to 1.) enhanced excitability of IP3Rs after IP3 binding; 2.) enhanced activity of store‐operated Ca2+ channels and T‐type voltage‐operated Ca2+ channels; 3.) decreased cytosolic Ca2+ removal due to inhibition of PMCA (plasma membrane Ca2+‐ATPase) and SERCA (sarco/endoplasmic reticulum Ca2+‐ATPase) calcium pumps. This type of calcium signal favors activation of the transcription factor NFAT (nuclear factor of activated T lymphocytes) that promotes hypertrophic growth and/or cell cycle progression. We suggest that the two main Ca2+‐regulated transcription factors, CREB and NFAT, exert opposite control over cell growth and/or proliferation. Therapeutic strategies based on lowering intracellular Ca2+ or targeting of Ca2+‐regulated transcription factors seems to be a promising approach to arrest growth and/or proliferation.

cAMP-Binding Protein Epac Induces Cardiomyocyte Hypertrophy

cAMP is one of the most important second messenger in the heart. The discovery of Epac as a guanine exchange factor (GEF), which is directly activated by cAMP, raises the question of the role of this protein in cardiac cells. Here we show that Epac activation leads to morphological changes and induces expression of cardiac hypertrophic markers. This process is associated with a Ca2+-dependent activation of the small GTPase, Rac. In addition, we found that Epac activates a prohypertrophic signaling pathway, which involves the Ca2+ sensitive phosphatase, calcineurin, and its primary downstream effector, NFAT. Rac is involved in Epac-induced NFAT dependent cardiomyocyte hypertrophy. Blockade of either calcineurin or Rac activity blunts the hypertrophic response elicited by Epac indicating these signaling molecules coordinately regulate cardiac gene expression and cellular growth. Our results thus open new insights into the signaling pathways by which cAMP may mediate its biological effects and identify Epac as a new positive regulator of cardiac growth.

Nonsynchronous accumulation of alpha-skeletal actin and beta-myosin heavy chain mRNAs during early stages of pressure-overload--induced cardiac hypertrophy demonstrated by in situ hybridization.

The development of cardiac hypertrophy secondary to pressure overload is accompanied by isoformic changes of contractile proteins such as myosin and actin. 35S-Labeled complementary RNA (cRNA) probes and in situ hybridization procedures were used for analysis of the regional distribution of newly formed transcripts from α-skeletal actin (α-sk-actin) and β-myosin heavy chain (β-MHC) genes during the early stages of pressure overload. The study was performed in 25-day-old rats submitted to a thoracic aortic stenosis and killed after surgery at times ranging from 4 hours to 3 days. Neither α-sk-actin nor β-MHC messenger RNA (mRNA) was detected in the hearts of normal and sham-operated animals. However, α-sk-actin mRNA accumulated throughout the entire left ventricle as early as 4 hours after aortic stenosis, and by 12 hours was also detected in the left atrium. In contrast, β-MHC mRNA was hardly detectable before day 1, and by days 2-3 was mainly restricted to the inner part of the left ventricle and around the coronary arteries. The absence of spatial and temporal coordination in the accumulation of α-sk-actin and β-MHC mRNAs indicates that different signals and/or regulatory mechanisms are implicated in the induction of the two genes in response to hemodynamic overload.

Ca2+ Cycling and New Therapeutic Approaches for Heart Failure

Received July 2, 2009; accepted October 5, 2009. Heart failure (HF) is a major health problem in Western countries. Despite significant progress in pharmacological and device-based treatment, the disease burden imposed continues to increase, particularly as the population ages. HF incidence approaches 10 per 1000 after age 65 years.1 Congestive HF is the final consequence of diverse cardiovascular disorders, including atherosclerosis, cardiomyopathy, and hypertension. Described as a complex pathophysiological syndrome that involves interactions of the circulatory, neurohormonal, and renal systems, HF is first a disease of the myocardium, although it soon induces defects in other systems. Current treatments for HF, focused on blocking neurohormonal pathways, improve survival, but they do not halt the progression of HF. Late-stage HF has a poor prognosis, and therapeutic options are limited. Faced with these challenges, researchers are exploring novel therapeutic options. Chronic HF is associated with increased sympathetic outflow, which may be compensatory early on, but long-term neurohormonal activation induces significant damage to the heart; in addition, it results in multiple alterations in the β-adrenergic receptor (β-AR) signaling cascade, including receptor downregulation, upregulation of receptor kinases, and increased inhibitory G-protein function.2 The amplitude and velocity of Ca2+ cycling are regulated by a dynamic balance of phosphorylation and dephosphorylation through kinases and phosphatases. Activation of β-ARs stimulates cAMP production and results in protein kinase A (PKA) phosphorylation of key regulators of excitation-contraction coupling, such as L-type Ca2+ channels, phospholamban, troponin I, ryanodine receptors (RyR), myosin-binding protein C, and protein phosphatase inhibitor-1 (I-1; Figure), which leads to increased amplitude and velocity of Ca2+ cycling and increased contractility on a beat-to-beat basis.3 Protein phosphatases PP1 and PP2A counterbalance phosphorylation of these proteins. There is clear evidence that alterations in sarcoplasmic reticulum (SR) Ca2+ cycling are a component of the impaired …

Multidrug resistance-associated protein 4 regulates cAMP-dependent signaling pathways and controls human and rat SMC proliferation

The second messengers cAMP and cGMP can be degraded by specific members of the phosphodiesterase superfamily or by active efflux transporters, namely the multidrug resistance-associated proteins (MRPs) MRP4 and MRP5. To determine the role of MRP4 and MRP5 in cell signaling, we studied arterial SMCs, in which the effects of cyclic nucleotide levels on SMC proliferation have been well established. We found that MRP4, but not MRP5, was upregulated during proliferation of isolated human coronary artery SMCs and following injury of rat carotid arteries in vivo. MRP4 inhibition significantly increased intracellular cAMP and cGMP levels and was sufficient to block proliferation and to prevent neointimal growth in injured rat carotid arteries. The antiproliferative effect of MRP4 inhibition was related to PKA/CREB pathway activation. Here we provide what we believe to be the first evidence that MRP4 acts as an independent endogenous regulator of intracellular cyclic nucleotide levels and as a mediator of cAMP-dependent signal transduction to the nucleus. We also identify MRP4 inhibition as a potentially new way of preventing abnormal VSMC proliferation.

Sarcoplasmic reticulum Ca 2+ ATPase as a therapeutic target for heart failure

The cardiac isoform of the sarco/endoplasmic reticulum Ca2+ATPase (SERCA2a) plays a major role in controlling excitation/contraction coupling. In both experimental and clinical heart failure, SERCA2a expression is significantly reduced which leads to abnormal Ca2+ handling and deficient contractility. A large number of studies in isolated cardiac myocytes and in small and large animal models of heart failure showed that restoring SERCA2a expression by gene transfer corrects the contractile abnormalities and improves energetics and electrical remodeling. Following a long line of investigation, a clinical trial is underway to restore SERCA2a expression in patients with heart failure using adeno-associated virus type 1. This review addresses the following issues regarding heart failure gene therapy: i) new insights on calcium regulation by SERCA2a; ii) SERCA2a as a gene therapy target in animal models of heart failure; iii) advances in the development of viral vectors and gene delivery; and iv) clinical trials on heart failure using SERCA2a. This review focuses on the new advances in SERCA2a- targeted gene therapy made in the last three years. In conclusion, SERCA2a is an important therapeutic target in various cardiovascular disorders. Ongoing clinical gene therapy trials will provide answers on its safety and applicability.

Sarco/Endoplasmic Reticulum Ca2+-ATPase Gene Transfer Reduces Vascular Smooth Muscle Cell Proliferation and Neointima Formation in the Rat

Proliferation of vascular smooth muscle cells (VSMC) is a primary cause of vascular disorders and is associated with major alterations in Ca2+ handling supported by loss of the sarco/endoplasmic reticulum calcium ATPase, SERCA2a. To determine the importance of SERCA2a in neointima formation, we have prevented loss of its expression by adenoviral gene transfer in a model of balloon injury of the rat carotid artery. Two weeks after injury, the intima/media ratio was significantly lower in SERCA2a-infected than in injured noninfected or injured &bgr;-galactosidase–infected carotids (0.29±0.04 versus 0.89±0.19 and 0.72±0.14, respectively; P<0.05), and was comparable to that observed in control carotids (0.21±0.03). The pathways leading to proliferation were analyzed in serum-stimulated VSMC. Forced expression of SERCA2a arrested cell cycle at the G1 phase and prevented apoptosis. SERCA2a inhibits proliferation through inactivation of calcineurin (PP2B) and its target transcription factor NFAT (nuclear factor of activated T-cells) resulting in lowering of cyclin D1 and pRb levels. By using NFAT-competing peptide VIVIT, we showed that NFAT activity is strongly required to promote VSMC proliferation. In conclusion, we provide the first evidence that increasing SERCA2a activity inhibits VSMC proliferation and balloon injury–induced neointima formation.