Phospholamban Expression Research Articles

Presenilin 1 mutation decreases both calcium and contractile responses in cerebral arteries

Mutations or upregulation in presenilin 1 (PS1) gene are found in familial early-onset Alzheimer's disease or sporadic late-onset Alzheimer's disease, respectively. PS1 has been essentially studied in neurons and its mutation was shown to alter intracellular calcium (Ca2+) signals. Here, we showed that PS1 is expressed in smooth muscle cells (SMCs) of mouse cerebral arteries, and we assessed the effects of the deletion of exon 9 of PS1 (PS1dE9) on Ca2+ signals and contractile responses of vascular SMC. Agonist-induced contraction of cerebral vessels was significantly decreased in PS1dE9 both in vivo and ex vivo. Spontaneous activity of Ca2+ sparks through ryanodine-sensitive channels (RyR) was unchanged, whereas the RyR-mediated Ca2+-release activated by caffeine was shorter in PS1dE9 SMC when compared with control. Moreover, PS1dE9 mutation decreased the caffeine-activated capacitive Ca2+ entry, and inhibitors of SERCA pumps reversed the effects of PS1dE9 on Ca2+ signals. PS1dE9 mutation also leads to the increased expression of SERCA3, phospholamban, and RyR3. These results show that PS1 plays a crucial role in the cerebrovascular system and the vascular reactivity is decreased through altered Ca2+ signals in PS1dE9 mutant mice.

Phospholamban is degraded through ubiquitin‐proteasome pathway by pVHL in failing hearts

The ubiquitin‐proteasome system (UPS) plays an essential and pivotal role for intracellular protein degradation and protein homeostasis. The von Hippel‐Lindau (VHL) is a tumor suppressor that forms E3 ubiquitin ligase complex, which negatively regulates the hypoxia‐inducible factor‐1a (HIF‐1a) by promoting its polyubiquitination. Although it is reported that protein VHL (pVHL) is involved in the stabilization of heart function via HIF1‐a regulation, the role of pVHL in UPS‐mediated protein degradation in the heart is not fully understood. Here, we show that phospholamban (PLN), which acts as a negative inhibitor of sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA2a), was co‐immunoprecipitated with pVHL in both PLN‐overexpressing HEK293 cells and mouse hearts with dilated cardiomyopathy (transgenic mice expressing an Arg9 to Cys point mutation in PLN, TgPLNR9C mice). Furthermore, the overexpression of VHL into HEK293 cells decreased PLN expression levels under the H2O2 stress condition, whereas knockdown of VHL by transient transfection with VHL‐siRNA increased PLN expression level both under the normal and the H2O2 stress conditions. Taken together with our previous study in which we showed that PLN is ubiquitinated at Lys3 residue, and polyubiquitination of PLN was significantly increased with its lower expression in TgPLNR9C mice compared to control, we propose that increased oxidative stress may increase the binding of pVHL with PLN to induce its degradation in failing hearts.

The Impact of Sevoflurane on Coupling of the Left Ventricular-to-Systemic Vasculature in Rats With Chronic Pulmonary Hypertension

The relationship between left ventricular function and afterload has not been investigated as much as the right ventricular function under chronic pulmonary hypertension (PH) during anesthesia. This study was designed to investigate effects of sevoflurane on the intrinsic coupling relationship between the left ventricle and systemic vasculature in the presence of PH. A randomized, controlled study. University hospital. Sprague-Dawley rats. Monocrotaline (MCT) was injected intraperitoneally to induce a PH model. Four weeks later, rats with MCT injection demonstrated significantly increased pulmonary arterial pressure and right/left ventricular systolic ratio of ventricular pressure (p < 0.001). Rats were treated with 1.5% sevoflurane inhalation. The PV catheters were inserted and left ventricular pressure-volume loops were measured at baseline, 30, 60, and 90 minutes during sevoflurane treatment. Preload recruitable stroke work and end-systolic elastance were decreased markedly in rats with MCT injection (p < 0.05). However, arterial elastance decreased similarly in both groups. Sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) level was decreased and the expression of phospholamban (PLB) was increased in the PH group and after sevoflurane treatment. PH rats suffered further SERCA2/PLB ratio decrease from their already low baseline. The left ventricular contractility and ventricular-vascular coupling were decreased in rats with PH after sevoflurane inhalation. Sevoflurane reduced SERCA2a expression and increased PLB expression in PH rats. This partially could explain why the LV contractility and ventricular-to-vasculature coupling of PH rats were attenuated after sevoflurane treatment.

Heart Failure Re-Distributes Phospholamban between Nuclear Membranes and Sarcoplasmic Reticulum in Cardiomyocytes

We recently documented that phospholamban (PLB), a sarcoplasmic reticulum (SR) resident protein regulator of cardiac SR Ca-ATPase (SERCA2a), is localized to the nuclear envelope (NE) of cardiomyocytes (CMs). It is well-known that heart failure (HF) remodeling involves altered protein expression of PLB and SERCA2a; however, with the demonstrated compartmentalization of PLB accumulation, we asked whether HF alters trafficking between ER/SR subcompartments affecting steady-state intracellular distribution of PLB and SERCA. In this study, we examined heart tissue from non-failing and pacing-induced HF dogs with the monoclonal PLB (2D12 or 1F1) and SERCA (2A7-A1) antibodies. Confocal immunofluorescence microscopy was used to compare subcellular concentration of PLB and SERCA. At least 3 regions from NE and SR in 10 CMs were analyzed for each dog, n = 4 dogs). Both PLB and SERCA antibodies stained SR and NE in these CMs. Fluorescence intensity ratios between NE and SR for PLB was 2.05±0.82 (mean ± SD) in control dog CMs, consistent with results just reported. In HF CMs we found a highly decreased ratio of PLB between NE and SR to 1.26 ± 0.34 (n = 8 dogs). In contrast to re-distribution of PLB, intensity ratios for SERCA in NE and SR remained unchanged (1.01±0.14 in Control, and 0.98±0.26 in HF). Furthermore, while there was only minor levels of phosphoPLB in Control CMs, we found that phosphoPLB (S16) were very significantly higher in NE of failed CMs. The decreased PLB relative to SERCA in the NE of CMs suggests that retention and accumulation of PLB in the NE is decreased in HF, leading to a smaller, possibly hyper-phosphorylated steady-state of PLB concentration in the NE.

Loss of myocardial retinoic acid receptor α induces diastolic dysfunction by promoting intracellular oxidative stress and calcium mishandling in adult mice

Retinoic acid receptor (RAR) has been implicated in pathological stimuli-induced cardiac remodeling. To determine whether the impairment of RARα signaling directly contributes to the development of heart dysfunction and the involved mechanisms, tamoxifen-induced myocardial specific RARα deletion (RARαKO) mice were utilized. Echocardiographic and cardiac catheterization studies showed significant diastolic dysfunction after 16wks of gene deletion. However, no significant differences were observed in left ventricular ejection fraction (LVEF), between RARαKO and wild type (WT) control mice. DHE staining showed increased intracellular reactive oxygen species (ROS) generation in the hearts of RARαKO mice. Significantly increased NOX2 (NADPH oxidase 2) and NOX4 levels and decreased SOD1 and SOD2 levels were observed in RARαKO mouse hearts, which were rescued by overexpression of RARα in cardiomyocytes. Decreased SERCA2a expression and phosphorylation of phospholamban (PLB), along with decreased phosphorylation of Akt and Ca2+/calmodulin-dependent protein kinase II δ (CaMKII δ) was observed in RARαKO mouse hearts. Ca2+ reuptake and cardiomyocyte relaxation were delayed by RARα deletion. Overexpression of RARα or inhibition of ROS generation or NOX activation prevented RARα deletion-induced decrease in SERCA2a expression/activation and delayed Ca2+ reuptake. Moreover, the gene and protein expression of RARα was significantly decreased in aged or metabolic stressed mouse hearts. RARα deletion accelerated the development of diastolic dysfunction in streptozotocin (STZ)-induced type 1 diabetic mice or in high fat diet fed mice. In conclusion, myocardial RARα deletion promoted diastolic dysfunction, with a relative preserved LVEF. Increased oxidative stress have an important role in the decreased expression/activation of SERCA2a and Ca2+ mishandling in RARαKO mice, which are major contributing factors in the development of diastolic dysfunction. These data suggest that impairment of cardiac RARα signaling may be a novel mechanism that is directly linked to pathological stimuli-induced diastolic dysfunction.

Mesenchymal stem cells suppress cardiac alternans by activation of PI3K mediated nitroso-redox pathway

BackgroundThe paracrine action of non-cardiac progenitor cells is robust, but not well understood. Mesenchymal stem cells (MSC) have been shown to enhance calcium (Ca++) cycling in myocytes. Therefore, we hypothesized that MSCs can suppress cardiac alternans, an important arrhythmia substrate, by paracrine action on Ca++ cycling. Methods and resultsHuman cardiac myocyte monolayers derived from iPS cells (hCM) were cultured without or with human MSCs (hMSC) directly or plated on a transwell insert. Ca++ transient alternans (Ca++ ALT) and Ca++ transient duration (CaD) were measured from hCM monolayers following application of 200μM H2O2. Ca++ ALT in hCM was significantly decreased when cultured with hMSCs directly (97%, p<0.0001) and when cultured with hMSC in the transwell insert (80%, p<0.0001). When hCM with hMSCs were pretreated with PI3K or eNOS inhibitors, Ca++ ALT was larger than baseline by 20% (p<0.0001) and 36% (p<0.0001), respectively. In contrast, Ca++ ALT was reduced by 89% compared to baseline (p<0.0001) when hCM monolayers without hMSCs were pretreated with 20μM GSNO. In all experiments, changes in Ca++ ALT were mirrored by changes in CaD. Finally, real time quantitative PCR revealed no significant differences in mRNA expression of RyR2, SERCA2a, and phospholamban between hCM cultured with or without hMSCs. ConclusionCa++ ALT is suppressed by hMSCs in a paracrine fashion due to activation of a PI3K-mediated nitroso-redox pathway. These findings demonstrate, for the first time, how stem cell therapy might be antiarrhythmic by suppressing cardiac alternans through paracrine action on Ca++ cycling.

Phospholamban degradation is induced by phosphorylation-mediated ubiquitination and inhibited by interaction with cardiac type Sarco(endo)plasmic reticulum Ca2+-ATPase

Phospholamban (PLN) regulates cardiac type sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA2a) via Ser16-phosphorylation. During heart failure, PLN expression is downregulated with SERCA2a; however, the mechanism of its regulation is not fully understood. Phosphorylation triggers protein degradation and because PLN phosphorylation is upregulated in failing hearts, we examined whether PLN is degraded by Ser16-phosphorylation. Cells overexpressing PLN exhibited its degradation post isoproterenol (Iso), forskolin, or 3-isobutyl-1-methylxanthine (IBMX) addition. Moreover, this degradation was inhibited by a cAMP-dependent protein kinase (PKA) inhibitor––H89. Co-immunoprecipitation revealed that Lys3 of PLN was oligo-ubiquitinated when ubiquitin was overexpressed, and was degraded by Iso treatment. However, when co-expressed with SERCA2a, oligo-ubiquitinated PLN at Lys3 was not degraded by Iso treatment. In failing hearts from 16 week-old TgPLNR9C mice, oligo-ubiquitinated PLN levels increased and PLN expression was downregulated. Furthermore, SERCA2a mRNA levels in TgPLNR9C mice hearts were lower than that in wild type mice; however, PLN mRNA levels showed no changes. In another heart failure model, MG132 treatment reversed PLN degradation. These data suggest that PLN is, at least partially, oligo-ubiquitinated at Lys3 and degraded through Ser16-phosphorylation-mediated poly-ubiquitination during heart failure.

Unique Localization of Phospholamban in Perinuclear Membranes of Cardiomyocytes from Several Species

Phospholamban (PLB) regulates sarcoplasmic reticulum (SR) calcium-ATPase (SERCA2a) in cardiomyocytes (CMs). In addition to their well-known location in SR membranes, only SERCA2a, but not PLB, is shown expressed in the perinuclear/nuclear membranes of CMs. Here we used confocal immunofluorescence microscopy to examine the intracellular distribution of PLB. As expected, in isolated mouse CMs, co-localized PLB and SERCA2a were detected in SR by the monoclonal antibodies 2D12 (against PLB) and 2A7-A1 (against SERCA2a), respectively. Importantly, in addition to 2A7-A1 binding signals, strong 2D12 binding signal was detected around the DAPI staining of the nuclei, indicating the expression of PLB in perinuclear/nuclear membranes. Further, we probed PLB expression in tissue sections from hearts of human, dog, or rabbit with use of a polyclonal (PLB9) and three monoclonal (2D12, 1G7, or 1F1) antibodies, which were produced against different epitope of PLB in a specie-dependent manner. In particular, 2D12 and PLB9 recognized all isoforms of PLB in these species and revealed PLB expression in SR as well as in the perinuclear/nuclear regions. In comparison, 1F1, the dog specie-specific antibody, recognized PLB expression in dog SR and in perinuclear/nuclear membranes, but is completely negative for human, mouse, or rabbit CMs. Moreover, 1G7, which is specie-specific for human, mouse, or rabbit isoform of PLB, recognized similar pattern of PLB expression in these species including in perinuclear membranes, but not in dog CMs. Finally, 2D12 binding signals were completely eliminated by co-incubation of a PLB peptide (residues 2-25). These results showed for the first time that in addition to its expression in SR, PLB is expressed in and around nuclear envelope of CMs.

Myocardial expression profiles of candidate molecules in patients with arrhythmogenic right ventricular cardiomyopathy/dysplasia compared to those with dilated cardiomyopathy and healthy controls

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is mainly an autosomal dominant disease characterized by fibrofatty infiltration of the right ventricle, leading to ventricular arrhythmias. Mutations in desmosomal proteins can be identified in about half of the patients. The pathogenic mechanisms leading to disease expression remain unclear. The purpose of this study was to investigate myocardial expression profiles of candidate molecules involved in the pathogenesis of ARVC/D. Myocardial messenger RNA (mRNA) expression of 62 junctional molecules, 5 cardiac ion channel molecules, 8 structural molecules, 4 apoptotic molecules, and 6 adipogenic molecules was studied. The averaged expression of candidate mRNAs was compared between ARVC/D samples (n = 10), nonfamilial dilated cardiomyopathy (DCM) samples (n = 10), and healthy control samples (n = 8). Immunohistochemistry and quantitative protein expression analysis were performed. Genetic analysis using next generation sequencing was performed in all patients with ARVC/D. Following mRNA levels were significantly increased in patients with ARVC/D compared to those with DCM and healthy controls: phospholamban (P ≤ .001 vs DCM; P ≤ .001 vs controls), healthy tumor protein 53 apoptosis effector (P = .001 vs DCM; P ≤ .001 vs controls), and carnitine palmitoyltransferase 1β (P ≤ .001 vs DCM; P = 0.008 vs controls). Plakophillin-2 (PKP-2) mRNA was downregulated in patients with ARVC/D with PKP-2 mutations compared with patients with ARVC/D without PKP-2 mutations (P = .04). Immunohistochemistry revealed significantly increased protein expression of phospholamban, tumor protein 53 apoptosis effector, and carnitine palmitoyltransferase 1β in patients with ARVC/D and decreased PKP-2 expression in patients with ARVC/D carrying a PKP-2 mutation. Changes in the expression profiles of sarcolemmal calcium channel regulation, apoptosis, and adipogenesis suggest that these molecular pathways may play a critical role in the pathogenesis of ARVC/D, independent of the underlying genetic mutations.

Abstract 16191: Phospholamban Regulates Perinuclear/nuclear Calcium Dynamics in Cardiomyocytes

Introduction: Phospholamban (PLB) regulates cardiac sarcoplasmic reticulum (SR) Ca 2+ -ATPase (SERCA2a), thus modulating SR Ca 2+ dynamics. Recent studies demonstrated that SERCA is involved in Ca 2+ uptake into the lumen of nuclear envelope (NE) of cardiomyocytes (CMs). However, the regulatory role of PLB on Ca 2+ uptake into NE remains unknown. Hypothesis: We hypothesize that PLB is also responsible for modulating nuclear Ca 2+ dynamics. Methods: Confocal immunofluorescence microscopy was used to determine subcellular expression of PLB. By using fluo-4 based confocal line-scan Ca 2+ imaging, we measured spontaneous Ca 2+ waves (SCWs) across both cytoplasmic and nuclear regions in isolated permeabilized mouse CMs. Results: Several anti-PLB antibodies strongly stained PLB at both SR and the perinuclear membranes in CMs. A PLB peptide (residues 1-31) eliminated all these anti-PLB antibody stains. To identify the functional role of PLB expressed in the perinuclear membranes, we took advantage of our recently established method that a Fab fragment of anti-PLB monoclonal antibody (Fab) reversed PLB inhibition specifically and increased SR Ca 2+ uptake and release. SCWs through the nuclear regions had typically relative low fluorescent amplitude (F/F 0 ) and slow decay time (t 1/2 ) compared to that in the cytoplasmic region. At the free intracellular Ca 2+ concentration ([Ca 2+ ] i ) of 400 nM, Fab (100 μg/mL) significantly enhanced F/F 0 and decreased t 1/2 of SCWs in both cytoplasmic and nuclear regions. After addition of Fab, F/F 0 of SCWs through the nuclear regions increased from 0.91±0.16 to 1.27±0.19 (n=9, p&lt;.05) while t 1/2 decreased from 137.6±18.5 ms to 105.0±11.3 ms, (p&lt;.05). Similar effects were also observed after phosphorylation of PLB by addition of 20 μM cAMP (F/F 0 =1.43±0.11 vs. 1.04±0.14 in control, p&lt;.05; t 1/2 =107.82±10.9 ms vs. 139.21±20.1 ms in control, n=6, p&lt;.05). At high [Ca 2+ ] i of 1000 nM where PLB does not inhibit SERCA2a, addition of cAMP or Fab had no significant effect on SCWs in both cytoplasmic and nuclear regions. Conclusions: We demonstrated that PLB is expressed in and around NE. Acute removal of PLB inhibition increased perinuclear/nuclear Ca 2+ uptake and release. PLB is critically involved in nuclear Ca 2+ signaling modulation.

A single resistance exercise session improves myocardial contractility in spontaneously hypertensive rats.

Resistance training evokes myocardial adaptation; however, the effects of a single resistance exercise session on cardiac performance are poorly understood or investigated. This study aimed to investigate the effects of a single resistance exercise session on the myocardial contractility of spontaneously hypertensive rats (SHRs). Male 3-month-old SHRs were divided into two groups: control (Ct) and exercise (Ex). Control animals were submitted to sham exercise. Blood pressure was measured in conscious rats before the exercise session to confirm the presence of arterial hypertension. Ten minutes after the exercise session, the animals were anesthetized and killed, and the hearts were removed. Cardiac contractility was evaluated in the whole heart by the Langendorff technique and by isometric contractions of isolated left ventricular papillary muscles. SERCA2a, phospholamban (PLB), and phosphorylated PLB expression were investigated by Western blot. Exercise increased force development of isolated papillary muscles (Ex=1.0±0.1 g/mg vs Ct=0.63±0.2 g/mg, P<0.05). Post-rest contraction was greater in the exercised animals (Ex=4.1±0.4% vs Ct=1.7±0.2%, P<0.05). Papillary muscles of exercised animals developed greater force under increasing isoproterenol concentrations (P<0.05). In the isolated heart, exercise increased left ventricular isovolumetric systolic pressure (LVISP; Δ +39 mmHg; P<0.05) from baseline conditions. Hearts from the exercised rats presented a greater response to increasing diastolic pressure. Positive inotropic intervention to calcium and isoproterenol resulted in greater LVISP in exercised animals (P<0.05). The results demonstrated that a single resistance exercise session improved myocardial contractility in SHRs.

Effects of Shenmai injection and its bioactive components following ischemia/reperfusion in cardiomyocytes.

The aim of the present study was to determine whether the myocardial protective function of Shenmai injection (SM) during ischemia/reperfusion (I/R) is attributable to its regulation of intracellular calcium (Ca2+) and phospholamban (PLB) levels. Cultured neonatal Sprague Dawley rat cardiomyocytes were used to compare the effects of normoxia, total saponins of Panax ginseng (TSPG), ginsenoside Rg1 (Rg1) and SM treatments in rat myocardial cells following I/R. For each of these treatment groups, the mRNA and protein levels of PLB and the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated, in addition to the cytoplasmic Ca2+ concentration [Ca2+]i and the rate of apoptosis. The results indicated that I/R markedly decreased phosphorylated PLB and SERCA expression and that SM was able to mitigate this effect, while TPSG and Rg1 were not. Furthermore, SM appeared to prevent aberrant apoptosis and restore the depleted [Ca2+]i resulting from I/R. The protective efficacy of SM against heart disease following I/R may, therefore, be due in part to its effect on intracellular Ca2+ homeostasis. SM may exert its protective effects by relieving PLB inhibition, and the pharmacodynamic actions of SM appear to be significantly more effective than those of its bioreactive components, TPSG and Rgl.

Atrial Fibrillation-Promoting Effect of Altered Left Atrial Calcium-Handling Proteins in Heart Failure

Background and Objectives: The incidence of atrial fibrillation (AF) is increased in heart failure (HF). Two major mechanisms of ventricular arrhythmia in HF are reentry mechanism due to tissue fibrosis and triggered activity due to an altered calcium-handling process. Unlike studies on ventricular arrhythmogenesis, only few studies investigated the relationship between changes in the calcium-handling process in atrial tissue and their relationship with atrial arrhythmogenesis in HF. Materials and Methods: HF was induced by myocardial infarction, and the rats were fed a normal sodium diet for 10 weeks and administered isoproterenol (30 μg/[kg∙h]) for the last 2 weeks of the experiment. Echocardiography and AF induction studies were conducted. In addition, mRNA and protein expressions of sarcoplasmic reticulum Ca 2+ -ATPase (SERCA), phospholamban (PLB), ryanodine receptor 2 (RyR2), and sodium calcium exchanger (NCX) were studied. Results: In the HF group, the ejection fractions were significantly decreased. AF duration (12.4±7.9 sec sham and 441.7±12.4 sec HF) and inducibility (8.0%±3.3% sham and 48.5%±10.6% HF) were significantly increased relative to the sham group. Atrial fibrosis tissue amounts were significantly increased in HF (0.20%±0.02% sham and 3.41%±0.45% HF). Atrial SERCA, PLB, RyR2 protein, and mRNA expressions were significantly decreased in HF, while the NCX protein expression was significantly increased in the HF group. The protein PLB/SERCA ratio was significantly increased by about 33% in the HF group. Conclusion: Changes in the protein and mRNA levels of calciumhandling proteins and increase in the degree of atrial fibrosis are important contributing factors of AF in severe HF.

PHD2/3-dependent hydroxylation tunes cardiac response to β-adrenergic stress via phospholamban.

Ischemic heart disease is the leading cause of heart failure. Both clinical trials and experimental animal studies demonstrate that chronic hypoxia can induce contractile dysfunction even before substantial ventricular damage, implicating a direct role of oxygen in the regulation of cardiac contractile function. Prolyl hydroxylase domain (PHD) proteins are well recognized as oxygen sensors and mediate a wide variety of cellular events by hydroxylating a growing list of protein substrates. Both PHD2 and PHD3 are highly expressed in the heart, yet their functional roles in modulating contractile function remain incompletely understood. Here, we report that combined deletion of Phd2 and Phd3 dramatically decreased expression of phospholamban (PLN), resulted in sustained activation of calcium/calmodulin-activated kinase II (CaMKII), and sensitized mice to chronic β-adrenergic stress-induced myocardial injury. We have provided evidence that thyroid hormone receptor-α (TR-α), a transcriptional regulator of PLN, interacts with PHD2 and PHD3 and is hydroxylated at 2 proline residues. Inhibition of PHDs increased the interaction between TR-α and nuclear receptor corepressor 2 (NCOR2) and suppressed Pln transcription. Together, these observations provide mechanistic insight into how oxygen directly modulates cardiac contractility and suggest that cardiac function could be modulated therapeutically by tuning PHD enzymatic activity.

Sex Hormones Promote Opposite Effects on ACE and ACE2 Activity, Hypertrophy and Cardiac Contractility in Spontaneously Hypertensive Rats.

BackgroundThere is growing interest in sex differences and RAS components. However, whether gender influences cardiac angiotensin I-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) activity is still unknown. In the present work, we determined the relationship between ACE and ACE2 activity, left ventricular function and gender in spontaneously hypertensive rats (SHRs).Methodology / Principal FindingsTwelve-week-old female (F) and male (M) SHRs were divided into 2 experimental groups (n = 7 in each group): sham (S) and gonadectomized (G). Fifty days after gonadectomy, we measured positive and negative first derivatives (dP/dt maximum left ventricle (LV) and dP/dt minimum LV, respectively), hypertrophy (morphometric analysis) and ACE and ACE2 catalytic activity (fluorimetrically). Expression of calcium handling proteins was measured by western blot. Male rats exhibited higher cardiac ACE and ACE2 activity as well as hypertrophy compared to female rats. Orchiectomy decreased the activity of these enzymes and hypertrophy, while ovariectomy increased hypertrophy and ACE2, but did not change ACE activity. For cardiac function, the male sham group had a lower +dP/dt than the female sham group. After gonadectomy, the +dP/dt increased in males and reduced in females. The male sham group had a lower -dP/dt than the female group. After gonadectomy, the -dP/dt increased in the male and decreased in the female groups when compared to the sham group. No difference was observed among the groups in SERCA2a protein expression. Gonadectomy increased protein expression of PLB (phospholamban) and the PLB to SERCA2a ratio in female rats, but did not change in male rats.ConclusionOvariectomy leads to increased cardiac hypertrophy, ACE2 activity, PLB expression and PLB to SERCA2a ratio, and worsening of hemodynamic variables, whereas in males the removal of testosterone has the opposite effects on RAS components.

Exposure to a Low Lead Concentration Impairs Contractile Machinery in Rat Cardiac Muscle.

Lead exposure has been considered to be a risk factor for hypertension and cardiovascular disease. Our purpose was to evaluate the effects of low plasma lead concentration on cardiac contractility in isolated papillary muscles. Wistar rats were divided in control group or group treated with 100 ppm of lead acetate in the drinking water for 15 days. Blood pressure (BP) was measured weekly. At the end of the treatment period, the animals were anesthetized and euthanized, and parameters related to isolated papillary muscle contractility were recorded. The lead concentrations in the blood reached 12.3 ± 2 μg/dL. The BP was increased in the group treated with 100 ppm of lead acetate. Lead treatment did not alter force and time derivatives of the force of left ventricular papillary muscles. In addition, the inotropic response induced by an increase in the extracellular Ca(2+) concentration was reduced in the Pb(2+) group. However, the uptake of Ca(2+) by the sarcoplasmic reticulum and the protein expression of SERCA and phospholamban remained unchanged. Postrest contraction was similar in the both groups, and tetanic peak and plateau tension were reduced in lead group. These results demonstrated that the reduction in the inotropic response to calcium does not appear to be caused by changes in the trans-sarcolemmal calcium flux but suggest that an impairment of the contractile machinery might be taking place. Our results demonstrate that even at a concentration below the limit considered to be safe, lead exerts deleterious effects on the cardiac contractile machinery.

Impaired oxidative metabolism and calcium mishandling underlie cardiac dysfunction in a rat model of post-acute isoproterenol-induced cardiomyopathy.

Stress-induced cardiomyopathy, triggered by acute catecholamine discharge, is a syndrome characterized by transient, apical ballooning linked to acute heart failure and ventricular arrhythmias. Rats receiving an acute isoproterenol (ISO) overdose (OV) suffer cardiac apex ischemia-reperfusion damage and arrhythmia, and then undergo cardiac remodeling and dysfunction. Nevertheless, the subcellular mechanisms underlying cardiac dysfunction after acute damage subsides are not thoroughly understood. To address this question, Wistar rats received a single ISO injection (67 mg/kg). We found in vivo moderate systolic and diastolic dysfunction at 2 wk post-ISO-OV; however, systolic dysfunction recovered after 4 wk, while diastolic dysfunction worsened. At 2 wk post-ISO-OV, cardiac function was assessed ex vivo, while mitochondrial oxidative metabolism and stress were assessed in vitro, and Ca(2+) handling in ventricular myocytes. These were complemented with sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), phospholamban (PLB), and RyR2 expression studies. Ex vivo, basal mechanical performance index (MPI) and oxygen consumption rate (MVO2) were unchanged. Nevertheless, upon increase of metabolic demand, by β-adrenergic stimulation (1-100 nM ISO), the MPI versus MVO2 relation decreased and shifted to the right, suggesting MPI and mitochondrial energy production uncoupling. Mitochondria showed decreased oxidative metabolism, membrane fragility, and enhanced oxidative stress. Myocytes presented systolic and diastolic Ca(2+) mishandling, and blunted response to ISO (100 nM), and all these without apparent changes in SERCA, PLB, or RyR2 expression. We suggest that post-ISO-OV mitochondrial dysfunction may underlie decreased cardiac contractility, mainly by depletion of ATP needed for myofilaments and Ca(2+) transport by SERCA, while exacerbated oxidative stress may enhance diastolic RyR2 activity.

Phospholamban phosphorylation, mutation, and structural dynamics: a biophysical approach to understanding and treating cardiomyopathy.

We review the recent development of novel biochemical and spectroscopic methods to determine the site-specific phosphorylation, expression, mutation, and structural dynamics of phospholamban (PLB), in relation to its function (inhibition of the cardiac calcium pump, SERCA2a), with specific focus on cardiac physiology, pathology, and therapy. In the cardiomyocyte, SERCA2a actively transports Ca2+ into the sarcoplasmic reticulum (SR) during relaxation (diastole) to create the concentration gradient that drives the passive efflux of Ca2+ required for cardiac contraction (systole). Unphosphorylated PLB (U-PLB) inhibits SERCA2a, but phosphorylation at S16 and/or T17 (producing P-PLB) changes the structure of PLB to relieve SERCA2a inhibition. Because insufficient SERCA2a activity is a hallmark of heart failure, SERCA2a activation, by gene therapy (Andino et al. 2008; Fish et al. 2013; Hoshijima et al. 2002; Jessup et al. 2011) or drug therapy (Ferrandi et al. 2013; Huang 2013; Khan et al. 2009; Rocchetti et al. 2008; Zhang et al. 2012), is a widely sought goal for treatment of heart failure. This review describes rational approaches to this goal. Novel biophysical assays, using site-directed labeling and high-resolution spectroscopy, have been developed to resolve the structural states of SERCA2a-PLB complexes in vitro and in living cells. Novel biochemical assays, using synthetic standards and multidimensional immunofluorescence, have been developed to quantitate PLB expression and phosphorylation states in cells and human tissues. The biochemical and biophysical properties of U-PLB, P-PLB, and mutant PLB will ultimately resolve the mechanisms of loss of inhibition and gain of inhibition to guide therapeutic development. These assays will be powerful tools for investigating human tissue samples from the Sydney Heart Bank, for the purpose of analyzing and diagnosing specific disorders.

Acute Doxorubicin-Induced Cardiotoxicity is Associated with Matrix Metalloproteinase-2 Alterations in Rats

Background: Doxorubicin can cause cardiotoxicity. Matrix metalloproteinases (MMP) are responsible for degrading extracellular matrix components which play a role in ventricular dilation. Increased MMP activity occurs after chronic doxorubicin treatment. In this study we evaluated in vivo and in vitro cardiac function in rats with acute doxorubicin treatment, and examined myocardial MMP and inflammatory activation, and gene expression of proteins involved in myocyte calcium transients. Methods: Wistar rats were injected with doxorubicin (Doxo, 20 mg/kg) or saline (Control). Echocardiogram was performed 48 h after treatment. Myocardial function was assessed in vitro in Langendorff preparation. Results: In left ventricle, doxorubicin impaired fractional shortening (Control 0.59±0.07; Doxo 0.51±0.05; p<0.001), and increased isovolumetric relaxation time (Control 20.3±4.3; Doxo 24.7±4.2 ms; p=0.007) and myocardial passive stiffness. MMP-2 activity, evaluated by zymography, was increased in Doxo (Control 141338 ± 8924; Doxo 188874 ± 7652 arbitrary units; p<0.001). There were no changes in TNF-α, INF-γ, IL-10, and ICAM-1 myocardial levels. Expression of phospholamban, Serca-2a, and ryanodine receptor did not differ between groups. Conclusion: Acute doxorubicin administration induces in vivo left ventricular dysfunction and in vitro increased myocardial passive stiffness in rats. Cardiac dysfunction is related to myocardial MMP-2 activation. Increased inflammatory stimulation or changed expression of the proteins involved in intracellular calcium transients is not involved in acute cardiac dysfunction.

Phospholamban as a crucial determinant of the inotropic response of human pluripotent stem cell-derived ventricular cardiomyocytes and engineered 3-dimensional tissue constructs.

Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs). However, a well-accepted roadblock has been their immature phenotype. hESC/iPSC-derived ventricular (v) CMs and their engineered cardiac microtissues (hvCMTs) similarly displayed positive chronotropic but null inotropic responses to β-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in β-adrenergic signaling, we investigated the functional consequences of PLB expression in hESC/iPSC-vCMs and hvCMTs. First, we confirmed that PLB protein was differentially expressed in hESC (HES2, H9)- and iPSC-derived and adult vCMs. We then transduced hES2-vCMs with the recombinant adenoviruses (Ad) Ad-PLB or Ad-S16E-PLB to overexpress wild-type PLB or the pseudophosphorylated point-mutated variant, respectively. As anticipated from the inhibitory effect of unphosphorylated PLB on sarco/endoplasmic reticulum Ca2+-ATPase, Ad-PLB transduction significantly attenuated electrically evoked Ca2+ transient amplitude and prolonged the 50% decay time. Importantly, Ad-PLB-transduced hES2-vCMs uniquely responded to isoproterenol. Ad-S16E-PLB-transduced hES2-vCMs displayed an intermediate phenotype. The same trends were observed with H9- and iPSC-vCMs. Directionally, similar results were also seen with Ad-PLB-transduced and Ad-S16E-transduced hvCMTs. However, Ad-PLB altered neither the global transcriptome nor ICa,L, implicating a PLB-specific effect. Engineered upregulation of PLB expression in hESC/iPSC-vCMs restores a positive inotropic response to β-adrenergic stimulation. These results not only provide a better mechanistic understanding of the immaturity of hESC/iPSC-vCMs but will also lead to improved disease models and transplantable prototypes with adult-like physiological responses.