Luteolin improves contractile function and attenuates apoptosis following ischemia–reperfusion in adult rat cardiomyocytes

AbstractIntracellular ion channel inositol 1,4,5-triphosphate receptor (IP3R1) releases Ca2+ from endoplasmic reticulum. The disturbance of IP3R1 is related to several neurodegenerative diseases. This study investigated the mechanism of IP3R1 in myocardial ischemia/reperfusion (MI/R). After MI/R modeling, IP3R1 expression was silenced in myocardium of MI/R rats to explore its role in the concentration of myocardial enzymes, infarct area, Ca2+ level, NLRP3/Caspase-1, and pyroptosis markers and inflammatory factors. The adult rat cardiomyocytes were isolated and cultured to establish hypoxia/reperfusion (H/R) cell model. The expression of IP3R1 was downregulated or ERP44 was overexpressed in H/R-induced cells. Nifedipine D6 was added to H/R-induced cells to block Ca2+ channel or Nigericin was added to activate NLRP3. IP3R1 was highly expressed in myocardium of MI/R rats, and silencing IP3R1 alleviated MI/R injury, reduced Ca2+ overload, inflammation and pyroptosis in MI/R rats, and H/R-induced cells. The binding of ERP44 to IP3R1 inhibited Ca2+ overload, alleviated cardiomyocyte inflammation, and pyroptosis. The increase of intracellular Ca2+ level caused H/R-induced cardiomyocyte pyroptosis through the NLRP3/Caspase-1 pathway. Activation of NLRP3 pathway reversed the protection of IP3R1 inhibition/ERP44 overexpression/Nifedipine D6 on H/R-induced cells. Overall, ERP44 binding to IP3R1 inhibits Ca2+ overload, thus alleviating pyroptosis and MI/R injury.

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Autophagy and protein kinase C are required for cardioprotection by sulfaphenazole

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00716.2009 ◽

2010 ◽

Vol 298 (2) ◽

pp. H570-H579 ◽

Cited By ~ 58

Author(s):

Chengqun Huang ◽

Wayne Liu ◽

Cynthia N. Perry ◽

Smadar Yitzhaki ◽

Youngil Lee ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Enhanced Recovery ◽

Ischemia Reperfusion ◽

Dominant Negative ◽

Rat Cardiomyocytes ◽

Adult Rat ◽

Kinase C ◽

Rat Hearts

Previously, we showed that sulfaphenazole (SUL), an antimicrobial agent that is a potent inhibitor of cytochrome P4502C9, is protective against ischemia-reperfusion (I/R) injury (Ref. 15 ). The mechanism, however, underlying this cardioprotection, is largely unknown. With evidence that activation of autophagy is protective against simulated I/R in HL-1 cells, and evidence that autophagy is upregulated in preconditioned hearts, we hypothesized that SUL-mediated cardioprotection might resemble ischemic preconditioning with respect to activation of protein kinase C and autophagy. We used the Langendorff model of global ischemia to assess the role of autophagy and protein kinase C in myocardial protection by SUL during I/R. We show that SUL enhanced recovery of function, reduced creatine kinase release, decreased infarct size, and induced autophagy. SUL also triggered PKC translocation, whereas inhibition of PKC with chelerythrine blocked the activation of autophagy in adult rat cardiomyocytes. In the Langendorff model, chelerythrine suppressed autophagy and abolished the protection mediated by SUL. SUL increased autophagy in adult rat cardiomyocytes infected with GFP-LC3 adenovirus, in isolated perfused rat hearts, and in mCherry-LC3 transgenic mice. To establish the role of autophagy in cardioprotection, we used the cell-permeable dominant-negative inhibitor of autophagy, Tat-Atg5K130R. Autophagy and cardioprotection were abolished in rat hearts perfused with recombinant Tat-Atg5K130R. Taken together, these studies indicate that cardioprotection mediated by SUL involves a PKC-dependent induction of autophagy. The findings suggest that autophagy may be a fundamental process that enhances the heart's tolerance to ischemia.

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Cardiac-targeted PIASy Gene Silencing Mediates deSUMOylation of Caveolin-3 and Prevents Ischemia/reperfusion-induced Nav1.5 Down-regulation and Ventricular Arrhythmias

10.21203/rs.3.rs-1163593/v1 ◽

2021 ◽

Author(s):

Chenchen Hu ◽

Xin Wei ◽

Jinmin Liu ◽

Linlin Han ◽

Chengkun Xia ◽

...

Keyword(s):

Gene Silencing ◽

Ventricular Arrhythmias ◽

Heart Diseases ◽

Ischemia Reperfusion ◽

Intraventricular Injection ◽

Membrane Expression ◽

Down Regulation ◽

Rat Cardiomyocytes ◽

Adult Rat

Abstract Background: Abnormal myocardial expression and function of Nav1.5 causes lethal ventricular arrhythmias during myocardial ischemia-reperfusion (I/R). PIASy mediated Caveolin-3 (Cav-3) SUMO modification affects Cav-3 binding to ligand Nav1.5. PIASy activity is increased after myocardial I/R, whether or not this may be attributable to plasma membrane Nav1.5 downregulation and ventricular arrhythmias remains unclear. Methods: Using recombinant adeno-associated virus subtype 9 (AAV9), rat cardiac PIASy was silenced by intraventricular injection of PIASy shRNA. Two weeks later, the hearts were subjected to I/R, and electrocardiography was performed to assess malignant arrhythmias. Tissues from peri-infarct areas of the left ventricle were collected for molecular biological measurement. Results: We found that PIASy was upregulated by I/R, with increased SUMO2/3 modification of Cav-3, reduced membrane Nav1.5 density, and increased ventricular arrhythmia frequency. These effects were significantly reversed by PIASy silencing. In addition, PIASy silencing enhanced Cav-3 binding to Nav1.5 and prevented I/R-induced Nav1.5 re-localization. Using in vitro models of HEK293T cells and isolated adult rat cardiomyocytes exposed to hypoxia/reoxygenation (H/R), this reserch further confirmed that PIASy promoted Cav-3 modification by SUMO2/3 and Nav1.5/Cav-3 dissociation after H/R. Mutation of the SUMO Consensus Sites Lysine in Cav-3 (K38R or K144R) alters the membrane expression levels of Nav1.5 and Cav-3 before and after H/R in HEK293T cells. Conclusions: I/R-induced cardiac PIASy activation contributes to Cav-3 SUMOylation by SUMO2/3 and dysregulated Nav1.5- related ventricular arrhythmias. Cardiac-targeted PIASy gene silencing mediates deSUMOylation of Cav-3 and prevents I/R-induced Nav1.5 down-regulation and ventricular arrhythmias in rats, identifying PIASy as a potential therapeutic target for relevant life-threatening arrhythmias in patients with ischemic heart diseases.

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Abstract 1120: Akt Modified MSCs Secrete a Novel Stem Cell Paracrine Factor that Protects Injured Heart, Activates Cyclin Dependent Kinase and Phosphorylates/Activates Akt Signaling Pathway in Adult Rat Cardiomyocytes

Circulation ◽

10.1161/circ.116.suppl_16.ii_225-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Jian Guo ◽

Maria Mirotsou ◽

Jing Huang ◽

Hui Mu ◽

Lunan Zhang ◽

...

Keyword(s):

Stem Cell ◽

Infarct Size ◽

Ischemia Reperfusion ◽

Cyclin Dependent Kinase ◽

Paracrine Factor ◽

Traditional Concept ◽

Rat Cardiomyocytes ◽

Intramyocardial Injection ◽

Adult Rat ◽

Novel Protein

Previously, we have shown that intracardiac injection of mesenchymal stem cell (MSC) overexpressing Akt (Akt-MSCs) could dramatically reduced infarct size and restored cardiac function in a paracrine manner. Microarray analysis of Akt-MSCs led to the identification of a novel transcript encoding a secreted protein. Cloning of the cDNA encoding this candidate gene into pET15b allowed the purification as a 6×His tagged recombinant protein. Interestingly, a 30 minute pre-incubation of the H9C2 myocytes with this protein (10nM) reduced H 2 O 2 induced apoptosis by 50% as measured by annexin V/PI staining on flow cytometry (n = 3, p<0.001). Moreover, this protein significantly prevent H 2 O 2 induced DNA fragmentation and inhibited Caspase 9 and Caspase 3/7 activities in adult rat cardiomyocytes by ~36% and ~42%, respectively (n=3, p<0.01). Furthermore, this protein dramatically inhibited the mitochondrial release of Cytochrome C and maintained mitochondrial Bcl-2 protein level. Intramyocardial injection of 1 μg of this novel protein dramatically reduced infarct size (−58%, n=10, p<0.001) and TUNEL positive nuclei (−69%, n=10, p<0.001) in a 30 min ischemia/24h reperfusion rat model. Fibrosis evidenced by collagen deposition was greatly reduced (−61%, n=8, p<0.001) 4 weeks after ischemia/reperfusion injury. Signaling studies demonstrated that this novel protein phosphorylated rat adult cardiomyocytes Akt Thr308 but not Akt Ser473 in a time-depended manner peaking at 30 min. Consequently, downstream Akt substrates, GSK3ß Ser9 and Bad Ser128 , are also phosphorylated at 30 min or at a later time point. In contrast to the traditional concept that PDK1 phosphorylates both Akt Thr308 and Akt Ser473 , our mass-spec and in vitro enzymatic analysis of rat cardiomyocytes stimulated with this factor suggests cyclin-dependent kinase 7 as being responsible for phosphorylation of Akt specifically at Thr 308 position. Further studies would be of great interest to interrogate whether this novel factor could activate other cyclin dependent kinase(s) and render the cardiomyocytes re-entering cell cycle. In conclusion, we have identified a novel paracrine factor from Akt-MSCs that mediates cardioprotective effects through activation of novel pathways involving Akt.

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Ischemic postconditioning and pinacidil suppress calcium overload in anoxia-reoxygenation cardiomyocytes via down-regulation of the calcium-sensing receptor

PeerJ ◽

10.7717/peerj.2612 ◽

2016 ◽

Vol 4 ◽

pp. e2612 ◽

Cited By ~ 6

Author(s):

Lin Zhang ◽

Song Cao ◽

Shengli Deng ◽

Gang Yao ◽

Tian Yu

Keyword(s):

Ischemia Reperfusion ◽

Calcium Overload ◽

Ischemic Postconditioning ◽

Free Calcium ◽

Protective Mechanism ◽

Calcium Sensing Receptor ◽

Down Regulation ◽

Rat Cardiomyocytes ◽

Adult Rat ◽

Calcium Sensing

Ischemic postconditioning (IPC) and ATP sensitive potassium channel (KATP) agonists (e.g. pinacidil and diazoxide) postconditioning are effective methods to defeat myocardial ischemia-reperfusion (I/R) injury, but their specific mechanisms of reducing I/R injury are not fully understood. We observed an intracellular free calcium ([Ca2+]i) overload in Anoxia/reoxygenation (A/R) cardiomyocytes, which can be reversed by KATP agonists diazoxide or pinacidil. The calcium-sensing receptor (CaSR) regulates intracellular calcium homeostasis. CaSR was reported to be involved in the I/R-induced apoptosis in rat cardiomyocytes. We therefore hypothesize that IPC and pinacidil postconditioning (PPC) reduce calcium overload in I/R cardiomyocytes by the down-regulation of CaSR. A/R model was established with adult rat caridomyocyte. mRNA and protein expression of CaSR were detected, IPC, PPC and KATP’s effects on [Ca2+]i concentration was assayed too. IPC and PPC ameliorated A/R insult induced [Ca2+]i overload in cardiomyocytes. In addition, they down-regulated the mRNA and protein level of CaSR as we expected. CaSR agonist spermine and KATP blocker glibenclamide offset IPC’s effects on CaSR expression and [Ca2+]i modulation. Our data indicate that CaSR down-regulation contributes to the mitigation of calcium overload in A/R cardiomyocytes, which may partially represents IPC and KATP’s myocardial protective mechanism under I/R circumstances.

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Abstract 38: A Decoy Peptide Selectively Inhibiting Dephosphorylation of Phospholamban

Circulation Research ◽

10.1161/res.111.suppl_1.a38 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Woo Jin Park ◽

Jae Gyun Oh ◽

Dongtak Jeong ◽

Roger J Hajjar

Keyword(s):

Ischemia Reperfusion ◽

Left Ventricular ◽

Cardiac Sarcoplasmic Reticulum ◽

Rat Cardiomyocytes ◽

Adult Rat ◽

Rat Hearts ◽

Cell Permeable Peptide ◽

Phosphorylated Peptide ◽

Developed Pressure

Cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) plays a crucial role in Ca2+ handling in cardiomyocytes. Phospholamban (PLB) is an endogenous inhibitor of SERCA2a and its inhibitory activity is enhanced by dephosphorylation by protein phosphatase 1 (PP1). Therefore, blocking PP1-mediated dephosphorylation of PLB would be an efficient strategy for restoration of the reduced SERCA2a activity in failing hearts. We sought to develop a decoy peptide that mimics the phosphorylated PLB and thus competitively inhibits the PP1-mediated dephosphorylation of PLB. The phosphorylation sites, Ser16 and Thr17, are located within the flexible extra-membrane loop (amino acids 14-22) of PLB. We therefore synthesized a 9-mer pseudo-phosphorylated peptide derived from this region with a replacement of Ser16 with Glu (ψ-PLB-SE). Two other 9-mer peptides with wild type PLB sequence (ψ-PLB) or with a replacement of Ser16 with Ala (ψ-PLB-SA) were also synthesized. These peptides were coupled to a cell-permeable peptide TAT to facilitate cellular uptake. Treatment of adult rat cardiomyocytes with TAT-ψ-PLB-SE, but not with TAT-ψ-PLB or TAT-ψ-PLB-SA, significantly elevated the phosphorylation level of PLB, concomitant with an increase in contractile parameters in vitro. Perfusion of isolated rat hearts with TAT-ψ-PLB-SE significantly restored the left ventricular developed pressure that was suppressed by ischemia-reperfusion (Fig. 1). These data indicate that ψ-PLB-SE prevented dephosphorylation of PLB by acting as a decoy for PP1 and it would provide effective modality to regulate SERCA2a activity in failing hearts.

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Distinct Roles for Sarcolemmal and Mitochondrial Adenosine Triphosphate-sensitive Potassium Channels in Isoflurane-induced Protection against Oxidative Stress

Anesthesiology ◽

10.1097/00000542-200607000-00018 ◽

2006 ◽

Vol 105 (1) ◽

pp. 98-104 ◽

Cited By ~ 37

Author(s):

Jasna Marinovic ◽

Zeljko J. Bosnjak ◽

Anna Stadnicka

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Adenosine Triphosphate ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Protective Mechanism ◽

Trypan Blue Exclusion ◽

Rat Cardiomyocytes ◽

Adult Rat ◽

Cardiac Preconditioning

Background Cardiac preconditioning, including that induced by halogenated anesthetics, is an innate protective mechanism against ischemia-reperfusion injury. The adenosine triphosphate-sensitive potassium (K(ATP)) channels are considered essential in preconditioning mechanism. However, it is unclear whether K(ATP) channels are triggers initiating the preconditioning signaling, and/or effectors responsible for the cardioprotective memory and activated during ischemia-reperfusion. Methods Adult rat cardiomyocytes were exposed to oxidative stress with 200 microM H(2)O(2) and 100 microM FeSO4. Myocyte survival was determined based on morphologic characteristics and trypan blue exclusion. To induce preconditioning, the myocytes were pretreated with isoflurane. The involvement of sarcolemmal and mitochondrial K(ATP) channels was investigated using specific inhibitors HMR-1098 and 5-hydroxydecanoic acid. Data are expressed as mean +/- SD. Results Oxidative stress induced cell death in 47 +/- 14% of myocytes. Pretreatment with isoflurane attenuated this effect to 26 +/- 8%. Blockade of the sarcolemmal K(ATP) channels abolished the protection by isoflurane pretreatment when HMR-1098 was applied throughout the experiment (50 +/- 21%) or only during oxidative stress (50 +/- 12%), but not when applied during isoflurane pretreatment (29 +/- 13%). Inhibition of the mitochondrial K(ATP) channels abolished cardioprotection irrespective of the timing of 5-hydroxydecanoic acid application. Cell death was 42 +/- 23, 45 +/- 23, and 46 +/- 22% when 5-hydroxydecanoic acid was applied throughout the experiment, only during isoflurane pretreatment, or only during oxidative stress, respectively. Conclusion The authors conclude that both sarcolemmal and mitochondrial K(ATP) channels play essential and distinct roles in protection afforded by isoflurane. Sarcolemmal K(ATP) channel seems to act as an effector of preconditioning, whereas mitochondrial K(ATP) channel plays a dual role as a trigger and an effector.

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In vivo adenoviral transfer of sorcin reverses cardiac contractile abnormalities of diabetic cardiomyopathy

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00245.2003 ◽

2004 ◽

Vol 286 (1) ◽

pp. H68-H75 ◽

Cited By ~ 36

Author(s):

Jorge Suarez ◽

Darrell D. Belke ◽

Bernd Gloss ◽

Thomas Dieterle ◽

Patrick M. McDonough ◽

...

Keyword(s):

Diabetic Cardiomyopathy ◽

Cardiac Myocyte ◽

Viral Vector ◽

Contractile Function ◽

Cardiac Contractility ◽

Rat Cardiomyocytes ◽

Adenoviral Gene Transfer ◽

Adult Rat ◽

Adenoviral Transfer

In many types of heart failure cardiac myocyte Ca2+ handling is abnormal because of downregulation of key Ca2+-handling proteins like sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a and ryanodine receptor (RyR)2. The alteration in SERCA2a and RyR2 expression results in altered cytosolic Ca2+ transients, leading to abnormal contraction. Sorcin is an EF-hand protein that confers the property of caffeine-activated intracellular Ca2+ release in nonmuscle cells by interacting with RyR2. To determine whether sorcin could improve the contractile function of the heart, we overexpressed sorcin in the heart of either normal or diabetic mice and in adult rat cardiomyocytes with an adenoviral gene transfer approach. Sorcin overexpression was associated with an increase in cardiac contractility of the normal heart and dramatically rescued the abnormal contractile function of the diabetic heart. These effects could be attributed to an improvement of the Ca2+ transients found in the cardiomyocyte after sorcin overexpression. Viral vector-mediated delivery of sorcin to cardiac myocytes is beneficial, resulting in improved contractile function in diabetic cardiomyopathy.

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Contractile Function is Altered by Regulation of HO-1 Activity in Single Adult Rat Cardiomyocytes

Biophysical Journal ◽

10.1016/j.bpj.2009.12.3943 ◽

2010 ◽

Vol 98 (3) ◽

pp. 720a

Author(s):

Sarah Nowakowski ◽

F. Steven Korte ◽

Jun Luo ◽

Margaret Allen ◽

Michael Regnier

Keyword(s):

Contractile Function ◽

Rat Cardiomyocytes ◽

Adult Rat

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Regulation of myocardial function by histidine-rich, calcium-binding protein

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01211.2003 ◽

2004 ◽

Vol 287 (4) ◽

pp. H1705-H1711 ◽

Cited By ~ 45

Author(s):

Guo-Chang Fan ◽

Kimberly N. Gregory ◽

Wen Zhao ◽

Woo Jin Park ◽

Evangelia G. Kranias

Keyword(s):

Heart Failure ◽

Ryanodine Receptor ◽

Calcium Binding ◽

Recombinant Adenovirus ◽

Contractile Function ◽

Binding Protein ◽

Human Heart Failure ◽

Rat Cardiomyocytes ◽

Adult Rat ◽

Protein Levels

Impaired sarcoplasmic reticulum (SR) Ca release has been suggested to contribute to the depressed cardiac function in heart failure. The release of Ca from the SR may be regulated by the ryanodine receptor, triadin, junctin, calsequestrin, and a histidine-rich, Ca-binding protein (HRC). We observed that the levels of HRC were reduced in animal models and human heart failure. To gain insight into the physiological function of HRC, we infected adult rat cardiac myocytes with a recombinant adenovirus that contains the full-length mouse HRC cDNA. Overexpression (1.7-fold) of HRC in adult rat cardiomyocytes was associated with increased SR Ca load (28%) but decreased SR Ca-induced Ca release (37%), resulting in impaired Ca cycling and depressed fractional shortening (36%) as well as depressed rates of shortening (38%) and relengthening (33%). Furthermore, the depressed basal contractile and Ca kinetic parameters in the HRC-infected myocytes remained significantly depressed even after maximal isoproterenol stimulation. Interestingly, HRC overexpresssion was accompanied by increased protein levels of junctin (1.4-fold) and triadin (1.8-fold), whereas the protein levels of ryanodine receptor, calsequestrin, phospholamban, and sarco(endo)plasmic reticulum Ca-ATPase remained unaltered. Collectively, these data indicate that alterations in expression levels of HRC are associated with impaired cardiac SR Ca homeostasis and contractile function.

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