Sarco/endoplasmic Reticulum Ca(2+) ATPase 2a Research Articles

Small molecule activator of Serca2a improves myocardial bioenergetics and lipid metabolism in diabetic mice

Background: Altered calcium homeostasis, insulin resistance, and dysregulated energy metabolism are major causes of diabetic cardiomyopathy. The expression and activity of Sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) are decreased in the diabetic heart failure. Increasing SERCA2a function reverses both systolic and diastolic dysfunction. However, the mechanisms and consequences underlying the role of SERCA2a in the diabetic failing heart are not clear yet. Objective: To examine the effect of pharmacological activation of SERCA2a on the diabetic heart. Methods and Results: To restore calcium abnormality in leptin-deficient (ob/ob) diabetic mice, we employed and contrasted two different models to elevate the restoration of SERCA2a function: 1) activation of SERCA2 with a novel allosteric agonist, CDN1163 or 2) overexpression of SERCA2a using cardiac-specific AAV9-mediated gene transfer. We demonstrate that SERCA2a activation with CDN1163 or AAV9 repressed the expression of genes involved in lipid synthesis and fatty acid oxidation in obese mice, leading to improved lipid homeostasis. Increased SERCA2a function improved mitochondrial function through elevation of mitochondrial biogenesis, mitochondrial DNA content, oxidative phosphorylation, and ATPase activity in the heart of obese mice. SERCA2a also restored mitochondrial membrane potential to normal levels in cultured H9C2 myocytes in the presence of high glucose and H2O2. SERCA2a ameliorated intracellular lipid toxicity and cell damage, possibly through the upregulation of the gene expression of antioxidant enzymes, and attenuated ROS generation by repression of NADPH oxidase in the obese heart. Furthermore, these molecular changes were paralleled by improvement in diabetes-induced diastolic dysfunction following CDN1163 activation of SERCA2a or AAV9-SERCA2a overexpression in the obese mice. Conclusion: These results suggest that calcium resolving diabetes-induced calcium dyshomeostasis by pharmacological activation of SERCA2a in obese individuals may be a highly effective therapeutic modality for treating obesity-induced diabetes. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cadmium exposure induces a sex-dependent decline in left ventricular cardiac function

AimsCadmium exposure is a worldwide problem that has been linked to the development of cardiovascular disease. This study aimed to elucidate mechanistic details of chronic cadmium exposure on the structure and function of the heart. Main methodsMale and female mice were exposed to cadmium chloride (CdCl2) via drinking water for eight weeks. Serial echocardiography and blood pressure measurements were performed. Markers of hypertrophy and fibrosis were assessed, along with molecular targets of Ca2+-handling. Key findingsMales exhibited a significant reduction in left ventricular ejection fraction and fractional shortening with CdCl2 exposure, along with increased ventricular volume at end-systole, and decreased interventricular septal thickness at end-systole. Interestingly, no changes were detected in females. Experiments in isolated cardiomyocytes revealed that CdCl2-induced contractile dysfunction was also present at the cellular level, showing decreased Ca2+ transient and sarcomere shortening amplitude with CdCl2 exposure. Further mechanistic investigation uncovered a decrease in sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein expression and phosphorylated phospholamban levels in male hearts with CdCl2 exposure. SignificanceThe findings of our novel study provide important insight into how cadmium exposure may act as a sex-specific driver of cardiovascular disease, and further underscore the importance of reducing human exposure to cadmium.

Identification of cell-biologic mechanisms of coronary artery spasm and its ex vivo diagnosis using peripheral blood-derived iPSCs

BackgroundAlthough vasospastic angina (VSA) is known to be caused by coronary artery spasm, no study has fully elucidated the exact underlying mechanism. Moreover, in order to confirm VSA, patients should undergo invasive coronary angiography with spasm provocation test. Herein, we investigated the pathophysiology of VSA using peripheral blood-derived induced pluripotent stem cells (iPSCs) and developed an ex vivo diagnostic method for VSA.Methods and resultsWith 10 mL of peripheral blood from patients with VSA, we generated iPSCs and differentiated these iPSCs into target cells. As compared with vascular smooth muscle cells (VSMCs) differentiated from iPSCs of normal subjects with negative provocation test, VSA patient-specific iPSCs-derived VSMCs showed very strong contraction in response to stimulants. Moreover, VSA patient-specific VSMCs exhibited a significant increase in stimulation-induced intracellular calcium efflux (Changes in the relative fluorescence unit [ΔF/F]; Control group vs. VSA group, 2.89 ± 0.34 vs. 10.32 ± 0.51, p < 0.01), and exclusively induced a secondary or tertiary peak of calcium efflux, suggesting that those findings could be diagnostic cut-off values for VSA. The observed hyperreactivity of VSA patient-specific VSMCs were caused by the upregulation of sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) due to its enhanced small ubiquitin-related modifier (SUMO)ylation. This increased activity of SERCA2a was reversed by treatment with ginkgolic acid, an inhibitor of SUMOylated E1 molecules (pi/µg protein; VSA group vs. VSA + ginkgolic acid, 52.36 ± 0.71 vs. 31.93 ± 1.13, p < 0.01).ConclusionsOur findings showed that abnormal calcium handling in sarco/endoplasmic reticulum could be induced by the enhanced SERCA2a activity in patients with VSA, leading to spasm. Such novel mechanisms of coronary artery spasm could be useful for drug development and diagnosis of VSA.

Reduced sarcoplasmic reticulum Ca2+ pump activity is anti-arrhythmic in ischemic cardiomyopathy

An arrhythmic mechanism seen only in heart failure (HF) that involves increased mitochondrial Ca2+ handling, selective transfer of Ca2+ to the sarcoplasmic reticulum (SR), and SR Ca2+ leak. Modeling suggests mitochondrial Ca2+ transfer to the SR via sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) is crucial to this arrhythmic mechanism. Here, we tested the role of SERCA2a in arrhythmias during ischemic HF. Myocardial infarction (MI) was induced in wild type (Wt) and SERCA2a heterozygous knockdown (SERCA+/-) mice.

Myeloid-Derived Growth Factor Protects Against Pressure Overload–Induced Heart Failure by Preserving Sarco/Endoplasmic Reticulum Ca 2+ -ATPase Expression in Cardiomyocytes

Background: Inflammation contributes to the pathogenesis of heart failure, but there is limited understanding of inflammation’s potential benefits. Inflammatory cells secrete MYDGF (myeloid-derived growth factor) to promote tissue repair after acute myocardial infarction. We hypothesized that MYDGF has a role in cardiac adaptation to persistent pressure overload. Methods: We defined the cellular sources and function of MYDGF in wild-type (WT), Mydgf -deficient ( Mydgf −/− ), and Mydgf bone marrow–chimeric or bone marrow–conditional transgenic mice with pressure overload–induced heart failure after transverse aortic constriction surgery. We measured MYDGF plasma concentrations by targeted liquid chromatography–mass spectrometry. We identified MYDGF signaling targets by phosphoproteomics and substrate-based kinase activity inference. We recorded Ca 2+ transients and sarcomere contractions in isolated cardiomyocytes. Additionally, we explored the therapeutic potential of recombinant MYDGF. Results: MYDGF protein abundance increased in the left ventricular myocardium and in blood plasma of pressure-overloaded mice. Patients with severe aortic stenosis also had elevated MYDGF plasma concentrations, which declined after transcatheter aortic valve implantation. Monocytes and macrophages emerged as the main MYDGF sources in the pressure-overloaded murine heart. While Mydgf −/− mice had no apparent phenotype at baseline, they developed more severe left ventricular hypertrophy and contractile dysfunction during pressure overload than WT mice. Conversely, conditional transgenic overexpression of MYDGF in bone marrow–derived inflammatory cells attenuated pressure overload–induced hypertrophy and dysfunction. Mechanistically, MYDGF inhibited G protein–coupled receptor agonist–induced hypertrophy and augmented SERCA2a (sarco/endoplasmic reticulum Ca 2+ -ATPase 2a) expression in cultured neonatal rat ventricular cardiomyocytes by enhancing PIM1 (Pim-1 proto-oncogene, serine/threonine kinase) expression and activity. Along this line, cardiomyocytes from pressure-overloaded Mydgf −/− mice displayed reduced PIM1 and SERCA2a expression, greater hypertrophy, and impaired Ca 2+ cycling and sarcomere function compared with cardiomyocytes from pressure-overloaded WT mice. Transplanting Mydgf −/− mice with WT bone marrow cells augmented cardiac PIM1 and SERCA2a levels and ameliorated pressure overload–induced hypertrophy and dysfunction. Pressure-overloaded Mydgf −/− mice were similarly rescued by adenoviral Serca2a gene transfer. Treating pressure-overloaded WT mice subcutaneously with recombinant MYDGF enhanced SERCA2a expression, attenuated left ventricular hypertrophy and dysfunction, and improved survival. Conclusions: These findings establish a MYDGF-based adaptive crosstalk between inflammatory cells and cardiomyocytes that protects against pressure overload–induced heart failure.

Pleiotropic, non-cell death-associated effects of inhibitors of receptor-interacting protein kinase 1 in the heart.

Inhibition of receptor-interacting protein kinase 1 (RIP1) has been recognized as a compelling tool for limiting necroptosis. Recent findings have indicated that RIP1 inhibitor, necrostatin-1 (Nec-1), is also able to modify heart function under non-cell death conditions. In this study, we investigated its underlying molecular mechanisms and compared with those of novel pharmacologically improved agents (Nec-1s and GSK'772) and its inactive analog (Nec-1i). Heart function was examined in Langendorff-perfused rat hearts. Certain proteins regulating myocardial contraction-relaxation cycle and oxidative stress (OS) were evaluated by immunoblotting and as the extent of lipid peroxidation, protein carbonylation and nitration, respectively. In spite of the increase of left ventricular developed pressure (LVDP) due to treatment by both Nec-1 and Nec-1i, only the former agent increased the phosphorylation of Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) at threonine 287 and cardiac myosin-binding protein-C (cMyBPc) at serine 282. In contrast, Nec-1s did not elicit such changes, while it also increased LVDP. GSK'772 activated CaMKIIδ-phospholamban (PLN) axis. Neither protein kinase A (PKA) nor its selected molecular targets, such as serine 16 phosphorylated PLN and sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a), were affected by either RIP1 inhibitor. Nec-1, like other necrostatins (Nec-1i, Nec-1s), but not GSK'772, elevated protein tyrosine nitration without affecting other markers of OS. In conclusion, this study indicated for the first time that Nec-1 may affect basal heart function by the modulation of OS and activation of some proteins of contraction-relaxation cycle.

Luteolin modulates SERCA2a via Sp1 upregulation to attenuate myocardial ischemia/reperfusion injury in mice

The sarco/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) is responsible for calcium transport during excitation–contraction coupling and is essential for maintaining myocardial systolic/diastolic function and intracellular Ca2+ levels. Therefore, it is important to investigate mechanisms whereby luteolin modulates SERCA2a expression to attenuate myocardial ischemia/reperfusion injury. C57BL/6j mice were randomly divided into eight groups. The expression and activity of SERCA2a was measured to assess interactions between the SERCA2a promoter and the Sp1 transcription factor, and the regulatory effects of luteolin. We used serum LDH release, serum cardiac troponin I level, hemodynamic data, myocardial infarction size and apoptosis-related indices to measure SERCA2a cardio-protective effects of luteolin pretreatment. Sp1 binding to SERCA2a promoter under ischemia/reperfusion conditions in the presence or absence of luteolin was analyzed by chromatin immunoprecipitation. Our experimental results indicated that during myocardial ischemia/reperfusion injury, luteolin pretreatment upregulated the expression levels of SERCA2a and Sp1. Sp1 overexpression enhanced the expression of SERCA2a at the transcriptional level. Luteolin pretreatment reversed the expression of SERCA2a through the increased expression of Sp1. Moreover, we demonstrated that luteolin pretreatment appeared to exert myocardial protective effects by upregulating the transcriptional activity of SERCA2a, via Sp1. In conclusion, during myocardial ischemia/reperfusion, Sp1 appeared to downregulate the expression of SERCA2a. Luteolin pretreatment was shown to improve SERCA2a expression via the upregulation of Sp1 to attenuate myocardial ischemia/reperfusion injury.

The Hypoxia Tolerance of the Goldfish (Carassius auratus) Heart: The NOS/NO System and Beyond.

The extraordinary capacity of the goldfish (Carassius auratus) to increase its cardiac performance under acute hypoxia is crucial in ensuring adequate oxygen supply to tissues and organs. However, the underlying physiological mechanisms are not yet completely elucidated. By employing an ex vivo working heart preparation, we observed that the time-dependent enhancement of contractility, distinctive of the hypoxic goldfish heart, is abolished by the Nitric Oxide Synthase (NOS) antagonist L-NMMA, the Nitric Oxide (NO) scavenger PTIO, as well as by the PI3-kinase (PI3-K) and sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) pumps’ inhibition by Wortmannin and Thapsigargin, respectively. In goldfish hearts exposed to hypoxia, an ELISA test revealed no changes in cGMP levels, while Western Blotting analysis showed an enhanced expression of the phosphorylated protein kinase B (pAkt) and of the NADPH oxidase catalytic subunit Nox2 (gp91phox). A significant decrease of protein S-nitrosylation was observed by Biotin Switch assay in hypoxic hearts. Results suggest a role for a PI3-K/Akt-mediated activation of the NOS-dependent NO production, and SERCA2a pumps in the mechanisms conferring benefits to the goldfish heart under hypoxia. They also propose protein denitrosylation, and the possibility of nitration, as parallel intracellular events.

Overexpression of SIRT3FL Improves Cardiac SERCA2a Function Following High Fat‐High Sucrose Feeding in Mice

BackgroundDiabetes induces a distinct form of cardiomyopathy, termed diabetic cardiomyopathy (DCM). A key pathophysiological mechanism of DCM is reduced level and activity of sarco(endo)plasmic reticulum Ca2+ ATPase 2a (SERCA2a). Recently, it has been discovered that the function of SERCA2a is negatively regulated by the post‐translational modification, acetylation. The sirtuin (SIRT) isoform, SIRT3 full‐length (SIRT3FL), deacetylates protein substrates, though it is unknown if SIRT3FL deacetylates SERCA2a. Therefore, the objective of this study was to determine if the overexpression of SIRT3FL alters cardiac SERCA2a acetylation and function in a mouse model of diabetes, induced by high fat‐high sucrose (HFHS) feeding.Methods and ResultsAdult (12 weeks of age) male C57BL/6J Wild Type (WT) mice and adult male MCK‐mSIRT3‐M1‐Flag transgenic (SIRT3FLTG) mice on a C57BL/6J background, who overexpress the SIRT3FL isoform in skeletal and cardiac muscle, were randomized into groups fed either a standard lab diet (Control) or a HFHS diet for 4‐months. After 4‐months, HFHS‐fed mice exhibited increased body weight, left ventricle (LV) weight, LV weight relative to body weight, and liver weight, compared to control fed‐mice. LV systolic parameters were unchanged by HFHS feeding, while the A and E wave forms of HFHS‐fed mice were slowed, compared to control‐fed mice. HFHS‐fed mice exhibited LV thickening, compared to control‐fed mice. Glucose tolerance was impaired in HFHS‐fed mice, compared to control‐fed mice. Diet did not alter SIRT3 protein content. Likewise, HFHS feeding did not alter the level or function of cardiac SERCA2a. LV SERCA2a protein content was not altered by genotype. However, SIRT3FLTG mice, regardless of diet, had 21% greater maximal SERCA activity (Vmax) and an 11% greater Hill coefficient, compared to WT mice. The Ca50, which is the Ca2+concentration that elicits 50% of Vmax, was 24% lower in SIRT3FLTG‐HFHS‐fed mice, compared to WT‐HFHS‐fed mice, indicating an increase in Ca2+ sensitivity.ConclusionHFHS feeding induced a diabetic phenotype in WT and SIRT3FLTG mice but level and function of cardiac SERCA2a was unchanged. However, overexpression of the SIRT3FL isoform improved cardiac SERCA2a function, regardless of HFHS feeding.Support or Funding InformationThis work was supported by a grant‐in‐aid from the Heart and Stroke Foundation of Canada

Nanoscale reorganization of sarcoplasmic reticulum in pressure-overload cardiac hypertrophy visualized by dSTORM

Pathological cardiac hypertrophy is a debilitating condition characterized by deleterious thickening of the myocardium, dysregulated Ca2+ signaling within cardiomyocytes, and contractile dysfunction. Importantly, the nanoscale organization, localization, and patterns of expression of critical Ca2+ handling regulators including dihydropyridine receptor (DHPR), ryanodine receptor 2 (RyR2), phospholamban (PLN), and sarco/endoplasmic reticulum Ca2+-ATPase 2A (SERCA2A) remain poorly understood, especially during pathological hypertrophy disease progression. In the current study, we induced cardiac pathological hypertrophy via transverse aortic constriction (TAC) on 8-week-old CD1 mice, followed by isolation of cardiac ventricular myocytes. dSTORM super-resolution imaging was then used to visualize proteins at nanoscale resolution at two time points and we quantified changes in protein cluster properties using Voronoi tessellation and 2D Fast Fourier Transform analyses. We showed a decrease in the density of DHPR and RyR2 clusters with pressure-overload cardiac hypertrophy and an increase in the density of SERCA2A protein clusters. PLN protein clusters decreased in density in 2-week TAC but returned to sham levels by 4-week TAC. Furthermore, 2D-FFT analysis revealed changes in molecular organization during pathological hypertrophy, with DHPR and RyR2 becoming dispersed while both SERCA2A and PLN sequestered into dense clusters. Our work reveals molecular adaptations that occur in critical SR proteins at a single molecule during pressure overload-induced cardiomyopathy. Nanoscale alterations in protein localization and patterns of expression of crucial SR proteins within the cardiomyocyte provided insights into the pathogenesis of cardiac hypertrophy, and specific evidence that cardiomyocytes undergo significant structural remodeling during the progression of pathological hypertrophy.

Programmed Cell Death 5 Provides Negative Feedback on Cardiac Hypertrophy Through the Stabilization of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2a Protein.

PDCD5 (programmed cell death 5) is ubiquitously expressed in tissues, including the heart; however, the mechanism underlying the cardiac function of PDCD5 has not been understood. We investigated the mechanisms of PDCD5 in the pathogenesis of cardiac hypertrophy. Cardiac-specific PDCD5 knockout mice developed severe cardiac hypertrophy and impaired cardiac function, whereas PDCD5 protein was significantly increased in transverse aortic constriction mouse hearts and phenylephrine-stimulated cardiomyocytes. Overexpression of PDCD5 inhibited phenylephrine-induced cardiomyocyte hypertrophy, and knockdown of PDCD5 induced cardiomyocyte hypertrophy and aggravated phenylephrine-induced hypertrophy. The expression of PDCD5 protein was regulated by NFATc2 (nuclear factor of activated T cells c2) during hypertrophy. SERCA2a (sarco/endoplasmic reticulum Ca2+-ATPase 2a) expression was decreased in PDCD5-deficient mouse hearts because of increased ubiquitination. PDCD5-deficient cardiomyocytes displayed decreased calcium uptake rate, slowed decay of Ca2+ transients, decreased calcium stores, and diastolic dysfunction. Moreover, reintroduction of PDCD5 in PDCD5-deficient mouse hearts reserved SERCA2a protein, suppressed NFATc2 protein, and rescued the hypertrophy and cardiac dysfunction. Our results revealed that PDCD5 is a novel target of NFATc2 in the hypertrophic heart and provides negative feedback to protect the heart against excessive hypertrophy via the stabilization of SERCA2a protein.

Abstract 570: Activin A Regulates SERCA2a Expression in Cardiomyocytes Through Ubiquitin-proteasome Mediated Degradation

Sarco/endoplasmic reticulum Ca 2+ -ATPase 2a (SERCA2a) is a critical regulator of cardiac function whose expression is decreased in aging and heart failure (HF). Activin-A, a member of the TGFβ superfamily, has been shown to be a potent paracrine inhibitor of SERCA2a expression in cardiomyocytes (CM). However, the mechanism by which this occurs remains unclear. Here, we confirm that Activin-A decreases SERCA2a protein expression in isolated CMs (reductions in SERCA2a expression at 25ng/ml: 35±5%, p=0.02; 50ng/ml: 55±14%, p= 0.003; 100ng/ml: 56±8%, p.= 0.001; n=2-3/group, three replicates). Moreover, we show that increasing circulating Activin-A levels in vivo also decreases cardiac SERCA2a protein (reductions in SERCA2a expression at 1-25ng/ml: 22±12%, p=0.27; 25-50ng/ml: 63±14%, p=0.0015; &gt;50ng/ml: 79±2%, p= 0.0002; n=4 per group). Using systematic gene set enrichment analysis of RNAseq data from hearts and CMs exposed to increased Activin-A, we identify the ubiquitin-proteasome system (UPS) as one of the most highly up-regulated pathways induced by Activin-A. With ubiquitination prediction modeling revealing multiple ubiquitin binding sites on SERCA2a, we hypothesized that Activin-A regulates SERCA2a expression through modulation of its UPS-mediated degradation. Indeed, inhibiting the proteasome with MG132 attenuated the inhibitory effects of Activin-A on CM SERCA2a expression. Although Activin-A did not directly affect proteasome activity in CMs, we found that it increased ubiquitination of SERCA2a, resulting in increased proteasome-mediated degradation. Additionally, targeted inhibition of the Activin type II receptors (ActRII) with a monoclonal ActRII blocking antibody reversed the effects of Activin-A on SERCA2a protein expression in isolated CMs in a dose-dependent fashion. Importantly, these effects of Activin-A/ActRII blockade on SERCA2a expression were recapitulated in vivo in a transverse aortic constriction model of HF, and further led to improvements in cardiac function. Collectively, these data identify a novel UPS-mediated mechanism by which Activin-A regulates CM SERCA2a expression, which warrants further investigation as a potential therapeutic approach to augmenting SERCA2a in HF.

The CCAAT box in the proximal SERCA2 gene promoter regulates basal and stress-induced transcription in cardiomyocytes.

The cardiac sarco/endoplasmic reticulum Ca2+-ATPase-2a (SERCA2a) is vital for the correct handling of calcium concentration in cardiomyocytes. Recent studies showed that the induction of endoplasmic reticulum (ER) stress (ERS) with the SERCA2 inhibitor Thapsigargin (Tg) increases the mRNA and protein levels of SERCA2a. The SERCA2 gene promoter contains an ERS response element (ERSE) at position -78bp that is conserved among species and might transcriptionally regulate SERCA2 gene expression. However, its involvement in SERCA2 basal and calcium-mediated transcriptional activation has not been elucidated. In this work, we show that in cellular cultures of neonatal rat ventricular myocytes, the treatment with Tg or the calcium ionophore A23187 increases the SERCA2a mRNA and protein abundance, as well as the transcriptional activity of two chimeric human SERCA2 gene constructs, containing -254 and -2579bp of 5'-regulatory region cloned in the pGL3-basic vector and transiently transfected in cultured cardiomyocytes. We found that the ERSE present in the SERCA2 proximal promoter contains a CCAAT box that is involved in basal and ERS-mediated hSERCA2 transcriptional activation. The EMSA results showed that the CCAAT box present in the ERSE recruits the NF-Y transcription factor. Additionally, by ChIP assays, we confirmed in vivo binding of NF-Y and C/EBPβ transcription factors to the SERCA2 gene proximal promoter.

Cardiovascular susceptibility to in vivo ischemic myocardial injury in male and female rat offspring exposed to prenatal hypoxia.

Intrauterine growth restriction (IUGR) following prenatal hypoxia exposure leads to a higher risk of developing cardiovascular disease (CVD) in later life. Our aim was to evaluate cardiac susceptibility and its pathophysiological mechanisms following acute myocardial infarction (MI) in adult rat offspring exposed to prenatal hypoxia. Male and female rat offspring, which experienced normoxia (21% O2) or hypoxia (11% O2) in utero underwent sham or MI surgery at 12 weeks of age. Echocardiographic data revealed that both sexes had systolic dysfunction following MI surgery, independent of prenatal hypoxia. Male offspring exposed to prenatal hypoxia, however, had left ventricular dilatation, global dysfunction, and signs of diastolic dysfunction following MI surgery as evident by increased left ventricular internal diameter (LVID) during diastole (MI effect, P<0.01), Tei index (MI effect, P<0.001), and E/E' ratio (prenatal hypoxia or MI effect, P<0.01). In contrast, diastolic dysfunction in female offspring was not as evident. Cardiac superoxide levels increased only in prenatal hypoxia exposed male offspring. Cardiac sarcoendoplasmic reticulum Ca2+-ATPase2a (SERCA2a) levels, a marker of cardiac injury and dysfunction, decreased in both male and female MI groups independent of prenatal hypoxia. Prenatal hypoxia increased cardiac ryanodine receptor 2 (RYR2) protein levels, while MI reduced RYR2 in only male offspring. In conclusion, male offspring exposed to prenatal hypoxia had an increased susceptibility to ischemic myocardial injury involving cardiac phenotypes similar to heart failure involving diastolic dysfunction in adult life compared with both offspring from healthy pregnancies and their female counterparts.

Istaroxime, a potential anticancer drug in prostate cancer, exerts beneficial functional effects in healthy and diseased human myocardium.

The current gold standard for prostate cancer treatment is androgen deprivation therapy and antiandrogenic agents. However, adverse cardiovascular events including heart failure can limit therapeutic use. Istaroxime, which combines Na+-K+-ATPase (NKA) inhibition with sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) stimulation, has recently shown promising anti-neoplastic effects in prostate cancer (PC) models and may also improve cardiac function. Considering the promising anticancer effects of istaroxime, we aimed to assess its functional effects on human myocardium.ResultsIstaroxime and strophanthidin elicited dose-dependent positive inotropic effects with a decline in developed force at supraphysiological concentrations in human atrial, nonfailing, and failing ventricular (ToF) myocardium. Diastolic force and RT50% did not change after exposure to both drugs. The maximal developed force in our in-vitro model of heart failure (ToF) was significantly higher after istaroxime administration. Such a difference did not occur in atrial or nonfailing ventricular trabeculae and was not applicable to the diastolic force.Materials and MethodsHuman atrial and ventricular trabeculae were isolated from nonfailing hearts and hearts of infants with tetralogy of Fallot (ToF), which were used as an in-vitro model of heart failure. The samples were electrically stimulated and treated with increasing concentrations of istaroxime and strophanthidin (10 nM–1 μM). Systolic and diastolic force development and relaxation parameters (RT50%) were analyzed.ConclusionsCombined NKA inhibition/SERCA2a stimulation increases contractility in atrial, nonfailing, and failing myocardium. Considering that heart failure is a potential side effect of current PC treatments, especially in elderly patients, istaroxime might combine beneficial cardiac and anti-cancer properties.

The function and significance of SERA2a in congestive heart failure: an analysis of gene therapy trials.

Congestive heart failure (CHF) is a widespread disease that has a negative impact on health, worldwide. Despite advances in therapies, morbidity, mortality and hospital discharges due to CHF remain high. Advances in the understanding of the pathophysiological mechanisms of CHF and the development of gene transfer technology have made gene therapy a realistic potential therapeutic method for CHF. Among the various potential targets, sarco-endoplasmic reticulum Ca²⁺-ATPase 2a (SERCA2a), which is an important protein in the regulation of Ca²⁺ cycling, has piqued the interest of many researchers. Restoring decreased SERCA2a activity in CHF could improve cardiac contractions and energetics, as well as reduce myocardial fibrosis and ventricular arrhythmias, and these benefits have been confirmed by studies using both in vivo and in vitro models. Following these promising preclinical results, SERCA2a gene therapy advanced to clinical trials. However, results of the clinical trials were controversial, leading some to question whether SERCA2a is the right target for CHF treatment. In this review, we illustrate the function and significance of SERCA2a in CHF, and more importantly, analyze possible causes of the controversial clinical trials results, with the aim of stimulating future research on the relationship between SERCA2a and CHF.

Loss of Rad-GTPase produces a novel adaptive cardiac phenotype resistant to systolic decline with aging.

Rad-GTPase is a regulator of L-type calcium current (LTCC), with increased calcium current observed in Rad knockout models. While mouse models that result in elevated LTCC have been associated with heart failure, our laboratory and others observe a hypercontractile phenotype with enhanced calcium homeostasis in Rad(-/-). It is currently unclear whether this observation represents an early time point in a decompensatory progression towards heart failure or whether Rad loss drives a novel phenotype with stable enhanced function. We test the hypothesis that Rad(-/-) drives a stable nonfailing hypercontractile phenotype in adult hearts, and we examine compensatory regulation of sarcoplasmic reticulum (SR) loading and protein changes. Heart function was measured in vivo with echocardiography. In vivo heart function was significantly improved in adult Rad(-/-) hearts compared with wild type. Heart wall dimensions were significantly increased, while heart size was decreased, and cardiac output was not changed. Cardiac function was maintained through 18 mo of age with no decompensation. SR releasable Ca(2+) was increased in isolated Rad(-/-) ventricular myocytes. Higher Ca(2+) load was accompanied by sarco/endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) protein elevation as determined by immunoblotting and a rightward shift in the thapsigargan inhibitor-response curve. Rad(-/-) promotes morphological changes accompanied by a stable increase in contractility with aging and preserved cardiac output. The Rad(-/-) phenotype is marked by enhanced systolic and diastolic function with increased SR uptake, which is consistent with a model that does not progress into heart failure.

MicroRNA therapy for the failing heart.

Heart failure (HF) may result from diverse pathological processes leading to progressive organ decompensation. Improper functionality of the calcium-handling machinery is a frequent component of the pathobiology underlying this debilitating process. Case in point, loss of intracellular calcium homeostasis because of decreased function of sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) is characteristic of HF, and restoration of SERCA2a function through gene therapy results in improved cardiac performance.1 MicroRNAs (miRNAs) constitute a new family of fine-tuning molecules involved in tight regulation of physiological and pathological processes. In fact, the identity and the level of miRNAs expressed at the systemic and organ levels change with disease. Wahlquist et al2 hypothesize that miRNAs capable of repressing contractility might be involved in HF and could, therefore, be used as therapeutic targets. Specifically, the authors focus their efforts on miRNAs involved in calcium dynamics regulation. To search for miRNAs capable of downregulating SERCA2a, a whole-genome collection of miRNAs was tested on Human Embryonic Kidney 293 cells cotransfected with an enhanced green fluorescent protein reporter fused to the 3′ untranslated region of the SERCA2A gene, enabling functional detection of miRNAs capable of decreasing SERCA2a through a fluorescence (enhanced green …

Blocking the urotensin II receptor pathway ameliorates the metabolic syndrome and improves cardiac function in obese mice

The metabolic syndrome is defined by the presence of hyperlipidemia, obesity, hypertension, and diabetes. The syndrome is associated with significant cardiovascular morbidity and mortality. The aim of the present study was to determine the role of the vasoactive peptide urotensin II (UII) in the pathogenesis of the metabolic syndrome. We used obese mice (ob/ob) to determine the effect of UII receptor (UT) blockage on the different facets of the metabolic syndrome with special emphasis on cardiac function. Our data demonstrate a significant increase in UII and UT expression in the myocardium of obese mice accompanied by a significant decrease in sarco/endoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) expression, as well as intracellular Na(+) and Ca(2+) compared with wild-type mice (P<0.05). Treatment of ob/ob mice with the UII receptor antagonist SB657510 significantly improved glucose levels, blood pressure, hyperlipidemia, expression of myocardial SERCA2a, intracellular Na(+) and Ca(2+) and cardiac function in association with a decrease in weight gain, and mammalian target of rapamycin (mTOR) and sodium/hydrogen exchanger 1 (NHE-1) protein expression compared with vehicle (P<0.05). These findings demonstrate an important role for UII in the pathogenesis of the metabolic syndrome and suggest that the use of UT receptor antagonists may provide a new therapeutic tool for the treatment of this syndrome.

ASSA13-03-16 Effect of b3-Adrenoceptor Signalling on Myocardial Fibrosis and Calciumion Regulation in Heart Failure

ObjectiveTo evaluate the effects of chronic β3-adrenoreceptor (β3-AR) agonism and antagonism on the expression of β3-AR in a isoproterenol (ISO)-induced heart failure (HF) rat model and to investigate the myocardial...