Sarcoplasmic Reticulum Calcium ATPase 2a Research Articles

Inhibition of miR-25 ameliorates cardiac and skeletal muscle dysfunction in aged mdx/utrn haploinsufficient (+/−) mice

Dystrophic cardiomyopathy is a significant feature of Duchenne muscular dystrophy (DMD). Increased cardiomyocyte cytosolic calcium (Ca2+) and interstitial fibrosis are major pathophysiological hallmarks that ultimately result in cardiac dysfunction. MicroRNA-25 (miR-25) has been identified as a suppressor of both sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) and mothers against decapentaplegic homolog-7 (Smad7) proteins. In this study, we created a gene transfer using an miR-25 tough decoy (TuD) RNA inhibitor delivered via recombinant adeno-associated virus serotype 9 (AAV9) to evaluate the effect of miR-25 inhibition on cardiac and skeletal muscle function in aged dystrophin/utrophin haploinsufficient mice mdx/utrn (+/-), a validated transgenic murine model of DMD. We found that the intravenous delivery of AAV9 miR-25 TuD resulted in strong and stable inhibition of cardiac miR-25 levels, together with the restoration of SERCA2a and Smad7 expression. This was associated with the amelioration of cardiomyocyte interstitial fibrosis as well as recovered cardiac function. Furthermore, the direct quadricep intramuscular injection of AAV9 miR-25 TuD significantly restored skeletal muscle Smad7 expression, reduced tissue fibrosis, and enhanced skeletal muscle performance in mdx/utrn (+/-) mice. These results imply that miR-25 TuD gene transfer may be a novel therapeutic approach to restore cardiomyocyte Ca2+ homeostasis and abrogate tissue fibrosis in DMD.

IL-37 Modulates Myocardial Calcium Handling via the p-STAT3/SERCA2a Axis in HF-Related Engineered Human Heart Tissue.

Interleukin-37 (IL-37) is a potent anti-inflammatory cytokine belonging to the IL-1 family. This study investigates the regulatory mechanism and reparative effects of IL-37 on HF-related human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) and engineered human heart tissue subjected to hypoxia and H2 O2 treatment. The contractile force and Ca2+ conduction capacity of the tissue are assessed using a stretching platform and high-resolution fluorescence imaging system. This investigation reveals that IL-37 treatment significantly enhances cell viability, calcium transient levels, contractile force, and Ca2+ conduction capacity in HF-related hiPSC-CMs and engineered human heart tissue. Notably, IL-37 facilitates the upregulation of sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) through enhancing nuclear p-STAT3 levels. This effect is mediated by the binding of p-STAT3 to the SERCA2a promoter, providing a novel insight on the reparative potential of IL-37 in HF. IL-37 demonstrates its ability to enhance systolic function by modulating myocardial calcium handling via the p-STAT3/SERCA2a axis in HF-related engineered human heart tissue (as shown in schematic diagram).

SENP2-mediated SERCA2a deSUMOylation increases calcium overload in cardiomyocytes to aggravate myocardial ischemia/reperfusion injury.

Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) is a key protein that maintains myocardial Ca 2+ homeostasis. The present study aimed to investigate the mechanism underlying the SERCA2a-SUMOylation (small ubiquitin-like modifier) process after ischemia/reperfusion injury (I/RI) in vitro and in vivo . Calcium transient and systolic/diastolic function of cardiomyocytes isolated from Serca2a knockout (KO) and wild-type mice with I/RI were compared. SUMO-relevant protein expression and localization were detected by quantitative real-time PCR (RT-qPCR), Western blotting, and immunofluorescence in vitro and in vivo . Serca2a-SUMOylation, infarct size, and cardiac function of Senp1 or Senp2 overexpressed/suppressed adenovirus infected cardiomyocytes, were detected by immunoprecipitation, triphenyltetrazolium chloride (TTC)-Evans blue staining, and echocardiography respectively. The results showed that the changes of Fura-2 fluorescence intensity and contraction amplitude of cardiomyocytes decreased in the I/RI groups and were further reduced in the Serca2a KO + I/RI groups. Senp1 and Senp2 messenger ribose nucleic acid (mRNA) and protein expression levels in vivo and in cardiomyocytes were highest at 6 h and declined at 12 h after I/RI. However, the highest levels in HL-1 cells were recorded at 12 h. Senp2 expression increased in the cytoplasm, unlike that of Senp1. Inhibition of Senp2 protein reversed the I/RI-induced Serca2a-SUMOylation decline, reduced the infarction area, and improved cardiac function, while inhibition of Senp1 protein could not restore the above indicators. I/RI activated Senp1 and Senp2 protein expression, which promoted Serca2a-deSUMOylation, while inhibition of Senp2 expression reversed Serca2a-SUMOylation and improved cardiac function.

The YAP/SERCA2a signaling pathway protects cardiomyocytes against reperfusion-induced apoptosis.

Mitochondria and the endoplasmic reticulum (ER) are known to promote cardiac ischemia/reperfusion (I/R) injury. Overexpression of yes-associated protein (YAP) and/or sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) has been shown to protect cardiomyocytes against I/R-induced injury. Here, we show that activation of the YAP/SERCA2a pathway attenuated mitochondrial damage and ER stress (ERS) to maintain cardiomyocyte viability in the setting of I/R injury. Our results demonstrate that I/R treatment reduced the transcription and expression of YAP and SERCA2a, along with a decline in cardiomyocyte viability. The overexpression of YAP promoted SERCA2a transcription, whereas SERCA2a upregulation did not affect the YAP transcription, suggesting that YAP functions upstream of SERCA2a. Activation of the YAP/SERCA2a pathway suppressed mitochondrial damage by sustaining the mitochondrial redox balance and restoring mitochondrial bioenergetics. Additionally, its activation repressed ERS, reduced calcium overload, and eventually blocked caspase activation. The knockdown of SERCA2a suppressed the protective effects of YAP overexpression on mitochondrial damage and ERS. Overall, our findings reveal that the YAP/SERCA2a pathway attenuates the mitochondrial damage and ERS in response to cardiac I/R injury by regulating the mitochondria–ER communication.

Diabetes mellitus exacerbates post-myocardial infarction heart failure by reducing sarcolipin promoter methylation.

AimsSarcolipin (SLN) is a key regulator of sarcoplasmic reticulum calcium‐ATPase (SERCA)2a, which handles intracellular calcium re‐uptake. This study was aimed to investigate the involvement of SLN in post‐myocardial infarction (MI) heart failure (HF) in diabetes.Methods and resultsDiabetes/MI rat models were established. Altered SLN expression in diabetic hearts was screened out by microarray. A myocardiotropic viral vector was used to deliver siRNA to silence SLN. DNA methylation was evaluated by bisulfite sequencing. Cardiac functions were evaluated by invasive haemodynamic examinations. The SERCA2a activity, cytoplasmic calcium concentration ([Ca2+]i), calcium spark, and myocyte contraction were detected. Correlation between HF and diabetes was analysed in a cohort consisted of 101 ST‐segment elevated myocardial infarction (STEMI) patients between 2017 and 2019 [53.54 ± 4.64 years old; 61.4% male gender; HbA1c% 6.15 ± 2.00; and left ventricular ejection fraction (LVEF%) 40.64 ± 3.20%]. SLN expression was evaluated in left ventricular tissue sample from six STEMI patients complicated with diabetes and six STEMI patients without diabetes. Expressions of DNA methyltransferase 1a and DNA methyltransferase 3 were reduced in diabetic hearts, leading to down‐regulation of SLN promoter methylation, resulting in increased SLN expression in rats. Impaired heart systolic functions were found in experimental diabetic MI rats, which were attenuated by SLN silencing. SERCA2a activity reduction and [Ca2+]i elevation were attenuated by SLN silencing in diabetic animal hearts and high‐glucose incubated primary myocytes. SLN silencing suppressed calcium sparks and improved contraction and sarcoplasmic reticulum calcium re‐uptake in high‐glucose incubated primary myocytes. Expression of SLN was up‐regulated in LV sampled from STEMI patients complicated with diabetes compared with non‐diabetic ones (P < 0.05). LVEF% was reduced in STEMI patients complicated with diabetes compared with non‐diabetic ones (P < 0.01). HbA1c% and LVEF% was related (r = −0.218, P = 0.028). Increased HbA1c% was correlated with reduced LVEF% after adjustment for age, sex, body mass index, cigarette smoking, creatinine, UA, low density lipoprotein, K+, Na+, and troponin I (adjusted odds ration = 0.75, 95% confidence interval 0.62–0.90, P = 0.002).ConclusionsDiabetes increases the vulnerability of STEMI patients to post‐MI HF by down‐regulating SLN promoter methylation, which further regulates SERCA2a activity via increasing cardiac SLN expression.

Circulating Sphingosine-1-Phosphate as A Non-Invasive Biomarker of Heart Transplant Rejection

Accumulating evidence has confirmed that the expression of sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) is downregulated in heart failure and cardiac allograft rejection. Although many SERCA2a-related genes and proteins involved in the regulation of myocardial Ca2+ fluxes have been explored, its related metabolites remain poorly studied. Our main objective was to identify circulating SERCA2a-related metabolites altered in cardiac allograft rejection and to determine whether these could serve as non-invasive biomarkers. Sixty plasma samples from adult heart transplant were included in a metabolomic analysis. Sphingosine-1 phosphate (S1P), metabolite closely related with SERCA, were increased in patients with cardiac rejection (p < 0.0001). S1P discriminated between patients with and without rejection: normal grafts vs. all rejecting grafts (AUC = 0.911, p < 0.0001), normal grafts vs. Grade 1 R (AUC = 0.819, p < 0.01), Grade 2 R (AUC = 0.911, p < 0.0001), Grade 3 R (AUC = 0.996, p < 0.0001). In addition, we found changes in key enzymes and receptors of S1P pathway analysed on explanted hearts from heart failure patients. This preliminary study reveals that circulating S1P determination could be a novel approach to detect cardiac rejection, showing a robust capability for detection that improves gradually with the severity of rejection. These alterations could be relevant to better understand the involvement of calcium regulation on the pathophysiology of rejection.

Long Noncoding RNA-DACH1 (Dachshund Homolog 1) Regulates Cardiac Function by Inhibiting SERCA2a (Sarcoplasmic Reticulum Calcium ATPase 2a).

Heart failure (HF) is a major cause of morbidity and mortality in patients with various cardiovascular diseases. Restoration of cardiac function is critical in improving the clinical outcomes of patients with HF. Long noncoding RNAs are widely involved in the development of multiple cardiac diseases, whereas their role in regulating cardiac function remains unclear. In this study, we found that the expression of long noncoding RNA-DACH1 (dachshund homolog 1) was upregulated in the failing hearts of mice and human. We tested the hypothesis that the intronic long noncoding RNA of DACH1 (LncDACH1) can participate in the regulation of cardiac function and HF. Transgenic overexpression of LncDACH1 in the cardiac myocytes of mice led to impaired cardiac function, reduced calcium transient and cell shortening, and decreased SERCA2a (sarcoplasmic reticulum calcium ATPase 2a) protein expression. In contrast, conditional knockout of LncDACH1 in cardiac myocytes resulted in increased calcium transient, cell shortening, SERCA2a protein expression, and improved cardiac function of transverse aortic constriction induced HF mice. The same qualitative data were obtained by overexpression or knockdown of LncDACH1 with adenovirus carrying LncDACH1 or its siRNA. Moreover, therapeutic administration of adenovirus carrying LncDACH1 siRNA to transverse aortic constriction mice abolished the development of HF. Mechanistically, LncDACH1 directly binds to SERCA2a. Overexpression of LncDACH1 augments the ubiquitination of SERCA2a. LncDACH1 upregulation impairs cardiac function by promoting ubiquitination-related degradation of SERCA2a.

[Expressions of SERCA2a and miR-25-3p/5p in myocardium of rats with heart failure and therapeutic effects of Xiefei Lishui recipe].

To investigate the expressions of sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) mRNA and miR-25-3p/5p in myocardium of rats with heart failure and the therapeutic effects of Xiefei Lishui recipe(Traditional Chinese Medicine). SD rats were randomly divided into five groups:control group, model group, Traditional Chinese Medicine group(TCM group), captopril group and digoxin group(n=10). Heart failure rat model was induced by intraperitoneal injections of doxorubicin(6 injections within 2 weeks; total dose:15 mg/kg). Five weeks after the first injection, Distilled water(10 ml/(kg·d)), TCM(22 g/(kg·d)), captopril(19 mg/(kg·d)),and digoxin(32μg/(kg·d))were administrated by gastrogavage for 35 days,respectively. Myocardial expressions of SERCA2a mRNA and miR-25-3p/5p were detected;myocardial activities of SERCA2a were measured; cardiac output(CO) and left ventricular ejection fraction(LVEF) and plasma level of B-type natriuretic peptide (BNP) were measured. Myocardial mRNA expression of SERCA2a was significantly down-regulated in rats with heart failure which could be significantly up-regulated by TCM or captopril or digoxin,while expressions of miR-25-3p and miR-25-5p in myocardium were significantly up-regulated which could be significantly down-regulated by TCM or captopril. SERCA2a activity, CO and LVEF in rats with heart failure were decreased significantly which could be significantly increased by TCM,while plasma level of BNP was increased significantly which could be significantly decreased by TCM or captopril or digoxin. CO in rats with heart failure could be signifi-cantly increased by captopril or digoxin. In rats with heart failure, myocardial mRNA expression of SERCA2a was downregulated, expression of miR-25-3p and miR-25-5p in myocardium was upregulated. Xiefei Lishui recipecan upregulate myocardial mRNA expression of SERCA2a, downregulate expression of miR-25-3p and miR-25-5p in myocardium and improve cardiac function.

Administration of anabolic steroid during adolescence induces long-term cardiac hypertrophy and increases susceptibility to ischemia/reperfusion injury in adult Wistar rats

Chronic administration of anabolic androgenic steroids (AAS) in adult rats results in cardiac hypertrophy and increased susceptibility to myocardial ischemia/reperfusion (IR) injury. Molecular analyses demonstrated that hyperactivation of type 1 angiotensin II (AT1) receptor mediates cardiac hypertrophy induced by AAS and also induces down-regulation of myocardial ATP-sensitive potassium channel (KATP), resulting in loss of exercise-induced cardioprotection. Exposure to AAS during adolescence promoted long-term cardiovascular dysfunctions, such as dysautonomia. We tested the hypothesis that chronic AAS exposure in the pre/pubertal phase increases the susceptibility to myocardial ischemia/reperfusion (IR) injury in adult rats. Male Wistar rats (26day old) were treated with vehicle (Control, n=12) or testosterone propionate (TP) (AAS, 5mgkg−1 n=12) 5 times/week during 5 weeks. At the end of AAS exposure, rats underwent 23days of washout period and were submitted to euthanasia. Langendorff-perfused hearts were submitted to IR injury and evaluated for mechanical dysfunctions and infarct size. Molecular analysis was performed by mRNA levels of α-myosin heavy chain (MHC), βMHC and brain-derived natriuretic peptide (BNP), ryanodine receptor (RyR2) and sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) by quantitative RT-PCR (qRT-PCR). The expression of AT1 receptor and KATP channel subunits (Kir6.1 and SURa) was analyzed by qRT-PCR and Western Blot. NADPH oxidase (Nox)-related reactive oxygen species generation was assessed by spectrofluorimetry. The expression of antioxidant enzymes was measured by qRT-PCR in order to address a potential role of redox unbalance. AAS exposure promoted long-term cardiac hypertrophy characterized by increased expression of βMHC and βMHC/αMHC ratio. Baseline derivative of pressure (dP/dt) was impaired by AAS exposure. Postischemic recovery of mechanical properties was impaired (decreased left ventricle [LV] developed pressure and maximal dP/dt; increased LV end-diastolic pressure and minimal dP/dt) and infarct size was larger in the AAS group. Catalase mRNA expression was significantly decreased in the AAS group. In conclusion, chronic administration of AAS during adolescence promoted long-term pathological cardiac hypertrophy and persistent increase in the susceptibility to myocardial IR injury possible due to disturbances on catalase expression.

Puerarin Enhances Ca2+ Reuptake and Ca2+ Content of Sarcoplasmic Reticulum in Murine Embryonic Stem Cell-Derived Cardiomyocytes via Upregulation of SERCA2a

Background/Aims: The embryonic stem cell-derived cardiomyocytes (ES-CMs) serve as potential sources for cardiac regenerative therapy. However, the immature sarcoplasmic reticulum (SR) function of ES-CMs prevents its application. In this report, we examined the effect of puerarin, an isoflavone compound, on SR function of murine ES-CMs. Methods: Murine ES-CMs were harvested by embryoid body-based differentiation method. Confocal calcium imaging and whole-cell patch clamps were performed to assess the function of SR. The mRNA expression levels of SR-related genes were examined by quantitative PCR. The protein expression of sarcoplasmic reticulum calcium-ATPase 2a (SERCA2a) was evaluated by immunofluorescent and western blot. Results: Long-term application of puerarin promotes basic properties of spontaneous calcium transient with increased amplitude, decay velocity, and decreased duration. Puerarin fails to alter I_Ca,L but increases the Ca²⁺ content of SR. Puerarin-treated ES-CMs have intact SR Ca²⁺ cycling with more SR Ca²⁺ reuptake. Long-term application of puerarin asynchronously upregulates the mRNA and protein expression of SERCA2a, as well as the transcripts of calsequestrin and triadin in developing ES-CMs. Application of puerarin during the stage of post-cardiac differentiation upregulates dose-dependently the transcripts of SERCA2a, phospholamban and tridin which can be reversed by the inhibitors of the PI3K/Akt and MAPK/ERK signaling pathways, but shows no effect on the protein expression of SERCA2a. Conclusion: This study demonstrates that long-term puerarin treatment enhances Ca²⁺ reuptake and Ca²⁺ content via upregulation of SERCA2a.

Exercise training restores the cardiac microRNA-1 and -214 levels regulating Ca2+ handling after myocardial infarction.

BackgroundImpaired cardiomyocyte contractility and calcium handling are hallmarks of left ventricular contractile dysfunction. Exercise training has been used as a remarkable strategy in the treatment of heart disease. The microRNA-1, which targets sodium/calcium exchanger 1 (NCX), and microRNA-214, which targets sarcoplasmic reticulum calcium ATPase-2a (Serca2a), are involved in cardiac function regulation. Thus, the aim of this study was to evaluate the effect of exercise training on cardiac microRNA-1 and −214 expression after myocardial infarction.MethodsWistar rats were randomized into four groups: sedentary sham (S-SHAM), sedentary infarction (S-INF), trained sham (T-SHAM), and trained infarction (T-INF). Exercise training consisted of 60 min/days, 5 days/week for 10 weeks with 3 % of body weight as overload beginning four weeks after myocardial infarction.ResultsMicroRNA-1 and −214 expressions were, respectively, decreased (52 %) and increased (54 %) in the S-INF compared to the S-SHAM, while exercise training normalized the expression of these microRNAs. The microRNA targets NCX and Serca-2a protein expression were, respectively, decreased (55 %) and increased (34 %) in the T-INF group compared to the S-INF group.ConclusionsThese results suggest that exercise training restores microRNA-1 and −214 expression levels and prevents change in both NCX and Serca-2a protein and gene expressions. Altogether, our data suggest a molecular mechanism to restore ventricular function after exercise training in myocardial infarction rats.

The Current and Future Landscape of SERCA Gene Therapy for Heart Failure: A Clinical Perspective

Gene therapy has been applied to cardiovascular disease for over 20 years but it is the application to heart failure that has generated recent interest in clinical trials. There is laboratory and early clinical evidence that delivery of sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy is beneficial for heart failure and this therapy could become the first positive inotrope with anti-arrhythmic properties. In this review we will discuss the rationale for SERCA2a gene therapy as a viable strategy in heart failure, review the published data, and discuss the ongoing clinical trials, before concluding with comments on the future challenges and potential for this therapy.

Cardiac tissue inhibitor of matrix metalloprotease 4 dictates cardiomyocyte contractility and differentiation of embryonic stem cells into cardiomyocytes: Road to therapy

BackgroundTIMP4 (Tissue Inhibitors of Matrix Metalloprotease 4), goes down in failing hearts and mice lacking TIMP4 show poor regeneration capacity after myocardial infarction (MI). This study is based on our previous observation that administration of cardiac inhibitor of metalloproteinase (~TIMP4) attenuates oxidative stress and remodeling in failing hearts. Therefore, we hypothesize that TIMP4 helps in cardiac regeneration by augmenting contractility and inducing the differentiation of cardiac progenitor cells into cardiomyocytes. MethodsTo validate this hypothesis, we transfected mouse cardiomyocytes with TIMP4 and TIMP4-siRNA and performed contractility studies in the TIMP4 transfected cardiomyocytes as compared to siRNA-TIMP4 transfected cardiomyocytes. We evaluated the calcium channel gene serca2a (sarcoplasmic reticulum calcium ATPase2a) and mir122a which tightly regulates serca2a to explain the changes in contractility. We treated mouse embryonic stem cells with cardiac extract and cardiac extract minus TIMP4 (using TIMP4 monoclonal antibody) to examine the effect of TIMP4 on differentiation of cardiac progenitor cells. ResultsContractility was augmented in the TIMP4 transfected cardiomyocytes as compared to siRNA-TIMP4 transfected cardiomyocytes. There was elevated expression of serca2a in the TIMP4 transformed myocytes and down regulation of mir122a. The cells treated with cardiac extract containing TIMP4 showed cardiac phenotype in terms of Ckit+, GATA4+ and Nkx2.5 expression. ConclusionThis is a novel report suggesting that TIMP4 augments contractility and induces differentiation of progenitor cells into cardiac phenotype. In view of the failure of MMP9 inhibitors for cardiac therapy, TIMP4 provides an alternative approach, being an indigenous molecule and a natural inhibitor of MMP9.

Abstract 241: Cardiac TIMP-4 Dictates cardiomyocyte contractility and differentiation of embryonic stem cells into cardiomyocytes: Road to Therapy

The remarkable nature of cardiomyocytes for contractility is attributed to the extracellular matrix which is maintained by the balance between MMPs (Matrix Metalloproteases) and TIMPs (Tissue Inhibitors of Matrix Metalloproteases). Any deviation from this delicate balance of MMP/TIMP is a hallmark of cardiovascular pathologies including myocardial infarction (MI). TIMP4, which is the least studied molecule, is deficient in failing hearts and mice lacking TIMP4 show poor regeneration capacity after MI. Therefore, we hypothesize that TIMP4 helps in cardiac regeneration by alleviating contractility and inducing the differentiation of cardiac progenitor cells into cardiomyocytes. To validate this hypothesis, we transfected cardiomyocytes with TIMP4 and TIMP4-siRNA and observed that there was increase in contractility in the TIMP4 transfected cardiomyocytes as compared to siRNA-TIMP4 transfected cardiomyocytes. To explain this we looked into the calcium channel genes and found increase in the expression of serca2a (sarcoplasmic reticulum calcium ATPase2a) in the TIMP4 transformed myocytes. Serca2a is tightly regulated by mir122a and we found decrease in the expression of mir122a in the TIMP4 transfected cells as compared to the TIMP4-siRNA cells. To observe the effect of TIMP4 in differentiation of cardiac progenitor cells we treated mouse embryonic stem cells with cardiac extract and cardiac extract minus TIMP4 (using TIMP4 monoclonal antibody). The cells treated with cardiac extract showed cardiac phenotype in terms of Ckit+, GATA4+ and Nkx2.5 expression. This is a novel report on the influence of TIMP4 on contractility and inducing the differentiation of stem cells to cardiomyocytes. In view of the failure of MMP9 inhibitors for cardiac therapy in clinical trials, TIMP4 provides and alternative approach, being an indigenous molecule, a natural inhibitor of MMP9 and efficient ROS scavenger.

Influence of araloside C on cardiac contractile function (850.7)

The present study aimed to determine the direct effect of araloside C on Ca2+ transient and contractions in isolated rat cardiomyocytes. In isolated adult rat cardiomyocytes, contractile and intracellular Ca2+ properties were determined simultaneously in real time by using an IonOptix MyoCam system. Western blot analysis was performed to evaluate the expressions of Ca2+ cycling proteins, including sarcoplasmic reticulum calcium ATPase 2a (SERCA2a), phospholamban (PLB), and Na+‐Ca2+ exchanger (NCX). Our results showed that araloside C directly increased sarcomere shortening, maximal velocity of shortening/relengthening (±dL/dt) and amplitude of [Ca2+]i transients in a concentration‐dependent manner. SERCA activity was also increased by araloside C. The expression of SERCA2a was not significantly upregulated, whereas PLB and NCX expressions were downregulated. These findings revealed the positive inotropic effect of araloside C on isolated rat cardiomyocytes. This effect was possibly associated with an increase in amplitude of the [Ca2+]i transient.Grant Funding Source: This study was supported by the National Natural Sciences Foundation of China (Grant No. 81173589).

Dipeptidylpeptidase Inhibition Is Associated with Improvement in Blood Pressure and Diastolic Function in Insulin-Resistant Male Zucker Obese Rats

Diastolic dysfunction is a prognosticator for future cardiovascular events that demonstrates a strong correlation with obesity. Pharmacological inhibition of dipeptidylpeptidase-4 (DPP-4) to increase the bioavailability of glucagon-like peptide-1 is an emerging therapy for control of glycemia in type 2 diabetes patients. Accumulating evidence suggests that glucagon-like peptide-1 has insulin-independent actions in cardiovascular tissue. However, it is not known whether DPP-4 inhibition improves obesity-related diastolic dysfunction. Eight-week-old Zucker obese (ZO) and Zucker lean rats were fed normal chow diet or diet containing the DPP-4 inhibitor, linagliptin (LGT), for 8 weeks. Plasma DPP-4 activity was 3.3-fold higher in ZO compared with Zucker lean rats and was reduced by 95% with LGT treatment. LGT improved echocardiographic and pressure volume-derived indices of diastolic function that were impaired in ZO control rats, without altering food intake or body weight gain during the study period. LGT also blunted elevated blood pressure progression in ZO rats involving improved skeletal muscle arteriolar function, without reducing left ventricular hypertrophy, fibrosis, or oxidative stress in ZO hearts. Expression of phosphorylated- endothelial nitric oxide synthase (eNOS)(Ser1177), total eNOS, and sarcoplasmic reticulum calcium ATPase 2a protein was elevated in the LGT-treated ZO heart, suggesting improved Ca(2+) handling. The ZO myocardium had an abnormal mitochondrial sarcomeric arrangement and cristae structure that were normalized by LGT. These studies suggest that LGT reduces blood pressure and improves intracellular Cai(2+) mishandling and cardiomyocyte ultrastructure, which collectively result in improvements in diastolic function in the absence of reductions in left ventricular hypertrophy, fibrosis, or oxidative stress in insulin-resistant ZO rats.

Chronic Therapy With a Partial Adenosine A1-Receptor Agonist Improves Left Ventricular Function and Remodeling in Dogs With Advanced Heart Failure

Adenosine elicits cardioprotection through A1-receptor activation. Therapy with adenosine A1-receptor agonists, however, is limited by undesirable actions of full agonism, such as bradycardia. This study examined the effects of capadenoson (CAP), a partial adenosine A1-receptor agonist, on left ventricular (LV) function and remodeling in dogs with heart failure. Twelve dogs with microembolization-induced heart failure were randomized to 12 weeks oral therapy with CAP (7.5 mg BID; n=6) or to no therapy (control; n=6). LV end-diastolic and end-systolic volumes, ejection fraction, plasma norepinephrine, and n-terminal pro-brain natriuretic peptide were measured before (pre) and 1 and 12 weeks after therapy (post). LV tissue obtained at post was used to assess volume fraction of interstitial fibrosis, sarcoplasmic reticulum calcium ATPase-2a activity, expression of mitochondria uncoupling proteins (UCP) and glucose transporters (GLUT). In controls, end-diastolic and end-systolic volumes increased and ejection fraction decreased significantly from pre to post (ejection fraction, 30±2 versus 27±1%; P<0.05). In CAP-treated dogs, end-diastolic volume was unchanged; ejection fraction increased significantly after 1 week (36±2 versus 27±2%; P<0.05) with a further increase at post (39±2%; P<0.05), whereas end-systolic volume decreased. CAP significantly decreased volume fraction of interstitial fibrosis, normalized sarcoplasmic reticulum calcium ATPase-2a activity and expression of UCP-2 and UCP-3, and GLUT-1 and GLUT-2 and significantly decreased plasma norepinephrine and n-terminal pro-brain natriuretic peptide. In heart failure dogs, CAP improves LV function and prevents progressive remodeling. Improvement of LV systolic function occurs early after initiating therapy. The results support development of partial adenosine A1-receptor agonists for the treatment of chronic heart failure.

Abstract 19538: Integrin-Linked Kinase Regulates Cardiomyocyte Calcium Signaling through Protein-Protein Interaction with Sarcoplasmic Reticulum Calcium ATPase

The transduction of mechanical load into dynamic changes in cardiac contractility is an incompletely understood process. ILK is a Ser/Thr kinase sarcomeric Z-disk protein with known mechanoreceptor function and cardioprotective properties. We hypothesized that ILK modulates calcium signaling in the cardiomyocyte to orchestrate both mechanoreceptor function and cytoprotection. ILK immunoprecipitates (IP) from transgenic mice with cardiac overexpression of ILK showed dose-dependent co-IP of sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) confirmed by mass spectroscopy. Transduction of wild type ILK upregulated SERCA2a expression, and increased the phosphorylation (Thr17) of phospholamban, in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). ILK knockdown by siRNA decreased SERCA2a expression. The upregulation of SERCA2a was not prevented by the ILK kinase small molecule inhibitor QLT0267 suggesting a scaffolding rather than catalytic effect of ILK. The beat rate response to the SERCA2a inhibitor thapsigargin (TG) in iPSC-CMs was determined by long-term measurements of electrical impedance using the xCELLigence RTCA Cardio System (Roche). ILK transduction resulted in a faster baseline beat rate compared to untreated and vector control cells (p &lt; 0.01). TG significantly decreased beat rate with incomplete recovery after 30 hours (p &lt; 0.01). TG also reduced beat rate in ILK-transduced cells; however, the recovery to pre-TG baseline was accelerated by the ILK transfection (time to recover 10 hr; p=0.017), consistent with increased ILK-mediated SERCA2a activity. Cardiomyocyte calcium transients were measured in human iPSC-CMs using confocal microscopy and analyzed using Origin 8.6. ILK-transduced iPSC-CMs showed a significantly reduced time constant Tau (p&lt;0.01) and increased beat rate (p=0.01) compared to GFP-reporter control, consistent with SERCA2a-dependent increased rate of SR-mediated calcium uptake. We conclude that ILK regulates the expression and activity of SERCA2a in the cardiomyocyte. The demonstrated ILK-SERCA2a interaction provides an elegant mechanism for transduction of mechano- and stress- reception into dynamic, calcium-mediated changes in cardiomyocyte contractility.

Influence of cyclovirobuxine D on intracellular [Ca2 +] regulation and the expression of the calcium cycling proteins in rat myocytes

Our previous studies have shown that cyclovirobuxine D (CVB-D) ameliorated the cardiac function in heart failure rats. Considering the relationship between cardiac function and [Ca2+]i, and the role of calcium cycling on regulating [Ca2+]i, the present study was designed to evaluate the influence of CVB-D on the calcium transient of myocytes from neonatal rats and adult heart failure (HF) rats. The expression of calcium cycling proteins, including L-type calcium channel (LTCC), ryanodine receptor 2 (RYR2), sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) and sodium–calcium exchanger (NCX), were investigated to explore the underlying mechanism. CVB-D increased the intensity of calcium transient, accelerated the process of calcium transient and attenuation in the neonatal and adult myocytes. Furthermore, CVB-D shortened Tpeak and Tattenuation in the adult myocytes and slowed down the heart rate of neonatal myocytes. Besides, CVB-D increased the expression of RYR2 and SERCA2a, decreased the expression of NCX, but showed no significant effect on LTCC. Thus, it was concluded that CVB-D increased the release and uptake of Ca2+ in systolic and diastolic period, respectively. CVB-D might not only facilitate the utilization of intracellular Ca2+, but also prevent the loss of Ca2+.

Immunosuppression decreases inflammation and increases AAV6-hSERCA2a-mediated SERCA2a expression.

The calcium pump SERCA2a (sarcoplasmic reticulum calcium ATPase 2a), which plays a central role in cardiac contraction, shows decreased expression in heart failure (HF). Increasing SERCA2a expression in HF models improves cardiac function. We used direct cardiac delivery of adeno-associated virus encoding human SERCA2a (AAV6-hSERCA2a) in HF and normal canine models to study safety, efficacy, and the effects of immunosuppression. Tachycardic-paced dogs received left ventricle (LV) wall injection of AAV6-hSERCA2a or solvent. Pacing continued postinjection for 2 or 6 weeks, until euthanasia. Tissue/serum samples were analyzed for hSERCA2a expression (Western blot) and immune responses (histology and AAV6-neutralizing antibodies). Nonpaced dogs received AAV6-hSERCA2a and were analyzed at 12 weeks; a parallel cohort received AAV-hSERCA2a and immunosuppression. AAV-mediated cardiac expression of hSERCA2a peaked at 2 weeks and then declined (to ~50%; p<0.03, 6 vs. 2 weeks). LV end diastolic and end systolic diameters decreased in 6-week dogs treated with AAV6-hSERCA2a (p<0.05) whereas LV diameters increased in control dogs. Dogs receiving AAV6-hSERCA2a developed neutralizing antibodies (titer ≥1:120) and cardiac cellular infiltration. Immunosuppression dramatically reduced immune responses (reduced inflammation and neutralizing antibody titers <1:20), and maintained hSERCA2a expression. Thus cardiac injection of AAV6-hSERCA2a promotes local hSERCA2a expression and improves cardiac function. However, the hSERCA2a protein level is reduced by host immune responses. Immunosuppression alleviates immune responses and sustains transgene expression, and may be an important adjuvant for clinical gene therapy trials.