Myocardial Protein Research Articles

CONTINUOUS EXPOSURE OF THE HYPERTENSIVE RATS TO LIGHT INCREASES THE RISK OF MALIGNANT CARDIAC ARRHYTHMIAS - OMACOR CARDIOPROTECTION

Objective: Light pollution disturbs circadian rhythm, and this can also be deleterious to the heart by increased susceptibility to arrhythmias. Herein, we investigated if rats exposed to continuous light had altered myocardial gene transcripts and/or protein expression which affects arrhythmogenesis. We then assessed if Omacorâ supplementation benefitted affected rats. Design and method: Male and female spontaneously hypertensive (SHR) and normotensive Wistar rats (WR) were housed under standard 12 h/12 h light/dark cycles or exposed to 6-weeks continuous 300 lux light for 24 h. Half the rats were then treated with 200 mg/100 g b.w. Omacorâ. Results: Continuous light resulted in higher male rat vulnerability to malignant ventricular fibrillation (VF). This was linked with myocardial connexin-43 (Cx43) down-regulation and deteriorated intercellular electrical coupling, due in part to increased pro-inflammatory NF-kB and iNOS transcripts and decreased sarcoplasmic reticulum Ca2+ATPase transcripts. Omacorâ treatment increased the electrical threshold to induce the VF linked with amelioration of myocardial Cx43 mRNA and Cx43 protein levels and the suppression of NF-kB and iNOS. This indicates that rat exposure to continuous light results in deleterious cardiac alterations jeopardizing intercellular Cx43 channel-mediated electrical communication, thereby increasing the risk of malignant arrhythmias. Conclusions: The adverse effects were attenuated by treatment with Omacorâ, thus supporting its potential benefit and the relevance of monitoring omega-3 index in human populations at risk. This research was supported by Slovak VEGA 2/0002/20, 2/0158/19, APVV 18-0548, 19-0317 and EU ITMS 26230120009.

SPONTANEOUSLY HYPERTENSIVE RATS EXHIBIT ENHANCED CARDIAC CONNEXIN-43 AND REDUCED PRO-FIBROTIC MARKERS IN RESPONSE TO COLD ACCLIMATION

Objective: We have previously shown that down-regulation of myocardial gap junction channel protein, connexin-43 (Cx43), and up-regulation of extracellular matrix (ECM) proteins in hearts of spontaneously hypertensive rats (SHR) promote occurrence of lethal arrhythmia. Cold acclimation has a potential for reducing cardiovascular risk, especially in setting of various pathological conditions, but there is a gap of knowledge concern the heart response to adaptive thermogenesis. The purpose of the study is to explore myocardial Cx43 and ECM proteins in genetically hairless strain of SHR (SHRM) that represents rewarding model of increased adaptive thermogenesis. Design and method: Adult hairless males and females SHRM strain that harbors mutated desmoglein-4 gene and their wild type SHR were housed at standard 22 C (that is below thermo-neutrality for these animals) and 12:12 hour light: dark cycle. WKY control rats were used as reference normotensive strain. Heart samples of the left ventricular tissue from euthanatized rats were used for the proteomics of Cx43, protein kinases, which phosphorylates Cx43, and for ECM markers TGF-, SMAD2/3, MMP-2, collagen-1. Immunofluorescence detection of Cx43 was performed to examine its myocardial topology. Results: Cx43 protein and its phosphorylated status was reduced in males and females SHR vs WKY rat hearts. In contrast, adaptive thermogenesis resulted in increased both parameters in SHRM regardless the sex. Moreover, pathological topology of Cx43 attributed to its enhanced localization at lateral sides of the cardiomyocytes in SHR was attenuated in SHRM. In addition, there were gentle differences in protein levels of PKA, PKG, PKC, MAPK, Akt between SHR and SHRM. Protein level of TGF- and SMAD 2/3 were increased in SHR compare to WKY rat hearts but decreased in SHRM unlike collagen-1 that was not altered in SHRM compare to SHR. Conclusions: Findings revealed beneficial up-regulation of myocardial Cx43 along with down-regulation of extracellular matrix proteins in response of hairless SHR to increased thermogenesis.

Effect of electroacupuncture preconditioning on expression of Gasdermin D, Caspase-1 and IL-1β in rats with myocardial ischemia reperfusion injury

To observe the effect of electroacupuncture(EA) preconditioning on expression of Caspase-1, Gasdermin D(GSDMD) and interleukin-1β(IL-1β) in myocardial tissue of myocardial ischemia reperfusion injury (MIRI) rats in order to explore its underlying mechanisms in resisting MIRI. Forty male rats were randomly divided into 4 groups: normal control (normal), sham operation (sham), MIRI model and EA groups. The MIRI model was established by ligation of the left anterior descending branch of the left coronary artery for 30 min and perfusion. EA (2 Hz/100 Hz, 1 mA) was applied to bilateral "Neiguan" (PC6) for 20 min, once a day for 3 consecutive days. The echocardiography was used to analyze the left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD) and left ventricular ejection fraction (LVEF, by using Teichholz formula) 4 h after modeling. The myocardial TTC staining was used to observe the proportion of the infarct area, and Western blot was used to detect the expression levels of GSDMD, Caspase-1, IL-1β proteins in the myocardium. Compared with the normal group, the immunoactivity of GSDMD was increased in the sham group (P<0.05). Compared with the sham group, the LVEF was significantly decreased (P<0.000 1), while the myocardial infarction area, immunoactivity of GSDMD, and the expression levels of Caspase-1, GSDMD and IL-1β proteins were considerably increased in the model group (P<0.000 1, P<0.001). In comparison with the model group, the decreased ejection fraction and the increased myocardial infarction area, and Caspase-1, GSDMD and IL-1β expression were reversed in the EA group (P<0.001, P<0.000 1, P<0.01). EA preconditioning may ameliorate myocardial injury in MIRI rats which may be associated with its function in down-regulating the expression of myocardial Caspase-1 protein to reduce cardiomyocyte pyroptosis.

Changes in AMPKα and Ubiquitin Ligases in Myocyte Reverse Remodeling after Surgical Ventricular Reconstruction in rats with ischemic cardiomyopathy.

The change in myocardial protein degradation systems after ventricular unloading has been unknown. We aimed to evaluate the anti-hypertrophic protein adenosine monophosphate-activated protein kinase (AMPK) and two major protein degradation systems (ubiquitin proteasome system and autophagy) in a model of surgical ventricular reconstruction (SVR) in rats with ischemic cardiomyopathy. Rats were randomized into the following groups: sham/sham (control group), myocardial infarction (MI)/sham (sham group) and MI/SVR (SVR group), with an interval of 4 weeks. Two (early, n = 5 for each) and 28 days (late, n = 5 for each) after SVR, ventricular size, and wall stress were assessed. Myocyte area, protein expression of AMPKα and autophagy markers, and gene expression of ubiquitin ligases (Atrogin-1 and Murf-1) were evaluated in the late phase. In the early phase, left ventricular dimensions and wall stress were smaller in the SVR group than in the sham group, whereas they were comparable in the late period. Myocyte area in the SVR group was reduced to the value in the control group, while it was larger in the sham group than in the control group. Total-AMPKα, p-AMPKα, and AMPKα phosphorylation rates were higher, and Atrogin-1 and Murf-1 were lower in the SVR group than in the sham group, while the autophagy markers were not different between the groups. p-AMPKα had strong negative correlations with myocyte area, Atrogin-1, and Murf-1. In myocyte reverse remodeling after SVR, AMPKα phosphorylation increased in association with reduced gene expression of ubiquitin ligases.

Protein kinase G phosphorylation of Chip protects ovariectomized females from myocardial infarction

Proteotoxicity from insufficient clearance of misfolded/damaged proteins exacerbates cardiac functional decline following a myocardial infarction (MI). Estrogen is known to play a crucial role in the survival rate of cardiomyocytes following a MI and is associated with increased protein degradation, however the mechanisms remain to be fully elucidated. Further estrogen has been shown to enhance myocardial protein kinase G (PKG) activity following an insult. In this study, we link this cardioprotective signaling pathway to the carboxyl terminus of Hsc70‐interacting protein (Chip), specifically via phosphorylation at serine 20 (pS20). Chip is a unique effector of proteostasisfunctioning as both a co‐chaperone and ubiquitin ligase. We test the role of Chip S20 with phospho‐mimetic (SE) and phospho‐null (SA) mice compared to WT mice in ovariectomized (OVX) and intact female mice following a MI. We did not observe anydifferences in cardiac function or proteostasis in sham mice regardless of genotype and OVX/intact surgery. Chip SE mice were protected against a MI in both OVX and intact female mice, as exhibited by maintained cardiac function and morphology. Chip WT, even moreso in Chip SA OVX mice, presented with decreased cardiac function, left ventricular chamber dilation and wall thinning, and myocardial fibrosis. Ubiquitinated proteins and protein aggregates were increased in the hearts of Chip SA OVX micewhile Chip SE OVX mice had little accumulation of either, both compared to Chip WT OVX mice. Notably the phospho‐status of Chip S20 did not alter the degradative capacities of the proteasome or lysosome, suggesting the underlying mechanism for enhanced proteostasis is due to increase functionality of the Chip complex. Collectively these dataindicate phosphorylation of Chip S20 is a critical regulator of cardiac proteostasis and is able to afford protection to OVX mice following a MI. These findings have clinical potential as post‐menopausal women are most at risk for deleterious outcomes following a heart attack.

Ubiquitin Carboxyl-Terminal Hydrolase L1 of Cardiomyocytes Promotes Macroautophagy and Proteostasis and Protects Against Post-myocardial Infarction Cardiac Remodeling and Heart Failure.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinase known to play essential roles in the nervous tissue. Myocardial upregulation of UCHL1 was observed in human dilated cardiomyopathy and several animal models of heart disease, but the (patho)physiological significance of UCHL1 in cardiomyocytes remains undefined. Hence, we conducted this study to fill this critical gap. We produced cardiomyocyte-restricted Uchl1 knockout (CKO) by coupling the Uchl1-floxed allele with transgenic Myh6-Cre in C57B/6J inbred mice. Mice transgenic for Myh6-Cre were used as controls (CTL). Myocardial Uchl1 proteins were markedly reduced in CKO mice but they did not display discernible abnormal phenotype. Ten-week old CTL or CKO mice were subjected to left anterior descending artery ligation (myocardial infarction, MI) or sham surgery (Sham) and characterized at 7- and 28-day after surgery. Compared with Sham mice, significant increases in myocardial UCHL1 proteins were detected in CTL MI but not in CKO MI mice. MI-induced left ventricular (LV) chamber dilation, reduction of ejection fraction (EF) and fractional shortening (FS), and LV anterior wall thinning detected by echocardiography were comparable between the CTL MI and CKO MI groups 7-day post-MI. However, by 28-day post-MI, MI-induced LV chamber dilatation, EF and FS reduction, increases of myocardial ubiquitin conjugates, and increases in the heart weight to body weight ratio and the ventricular weight to body weight ratio were significantly more pronounced in CKO MI than CTL MI mice. As further revealed by LV pressure-volume relationship analyses, CKO MI mice but not CTL MI mice displayed significant decreases in stroke volume, cardiac output, and the maximum rates of LV pressure rising or declining and of LV volume declining, as well as significant increases in LV end-diastolic pressure and Tau, compared with their respective Sham controls. LC3-II flux assays reveal that autophagic flux is decreased in CKO mouse myocardium as well as in cultured Uchl1-deficient cardiomyocytes. In conclusion, UCHL1 of cardiomyocytes is dispensable for development but promotes macroautophagy in cardiomyocytes. Upregulation of UCHL1 in post-MI hearts occurs primarily in the cardiomyocytes and protects against post-MI cardiac remodeling and malfunction likely through supporting autophagic flux and proteostasis during a stress condition.

QiShenYiQi pill for myocardial collagen metabolism and apoptosis in rats of autoimmune cardiomyopathy

Context QiShenYiQi pill (QSYQ) is a traditional Chinese medicine with a myocardial protective effect. Objective To explore the effect of QSYQ on myocardial collagen metabolism in rats with autoimmune cardiomyopathy and explore the underlying mechanism from the aspect of apoptosis. Materials and methods We established an autoimmune cardiomyopathy model using Lewis rats. The rats were then randomly divided into six groups (n = 8): control, model, 3-methyladenine (15 mg/kg, intraperitoneal injection), QSYQ low-dose (135 mg/kg, gavage), QSYQ medium dose (270 mg/kg, gavage), and QSYQ high-dose (540 mg/kg, gavage) for four weeks. Van Gieson staining was applied for myocardial pathological characteristics, TUNEL fluorescence for myocardial cell apoptosis, enzyme-linked immunosorbent assay (ELISA) for serum PICP, PIIINP, and CTX-I levels, and western blot analysis for type I/III myocardial collagen, Bcl-2, Bax, and caspase-3 proteins. Results Results showed that QSYQ (135, 270, or 540 mg/kg) significantly reduced the expression of myocardial type I/III collagen, and concentrations of serum PICP, PIIINP, and CTX-I in rats. Moreover, QSYQ could alleviate myocardial fibrosis more effectively at a higher dose. QSYQ could also inhibit myocardial apoptosis via downregulating Bcl-2 expression, and upregulating Bax and caspase-3 expression levels. Discussion and conclusions The QSYQ can improve myocardial collagen metabolism by inhibiting apoptosis, which provides a potential therapeutic approach for autoimmune cardiomyopathy.

Female hormones prevent sepsis-induced cardiac dysfunction: an experimental randomized study

Although epidemiologic research has demonstrated significant differences in incidence and outcomes of sepsis according to sex, their underlying biological mechanisms are poorly understood. Here, we studied the influence of hormonal status by comparing in vivo cardiac performances measured by MRI in non-ovariectomized and ovariectomized septic female rats. Control and ovariectomized rats were randomly allocated to the following groups: sham, sepsis and sepsis plus landiolol. Sepsis was induced by caecum ligation and punction (CLP). Landiolol, a short-acting selective β1-adrenergic blocker improving the in vivo cardiac performance of septic male rats was perfused continuously after sepsis induction. Cardiac MRI was carried out 18 h after induction of sepsis to assess in vivo cardiac function. Capillary permeability was evaluated by Evans Blue administration and measurement of its tissue extravasation. Variation in myocardial gene and protein expression was also assessed by qPCR and western-blot in the left ventricular tissue. Sepsis reduced indexed stroke volume, cardiac index and indexed end-diastolic volume compared to sham group in ovariectomized females whereas it had no effect in control females. This was associated with an overexpression of JAK2 expression and STAT3 phosphorylation on Ser727 site, and an inhibition of the adrenergic pathways in OVR females. Landiolol increased the indexed stroke volume by reversing the indexed end-diastolic volume reduction after sepsis in ovariectomized females, while it decreased indexed stroke volume and cardiac index in control. This was supported by an overexpression of genes involved in calcium influx in OVR females while an inactivation of the β-adrenergic and a calcium efflux pathway was observed in control females. Sepsis decreased in vivo cardiac performances in ovariectomized females but not in control females, presumably associated with a more pronounced inflammation, inhibition of the adrenergic pathway and calcium efflux defects. Administration of landiolol prevents this cardiac dysfunction in ovariectomized females with a probable activation of calcium influx, while it has deleterious effects in control females in which calcium efflux pathways were down-regulated.

Effect of Electroacupuncture at Wushu Acupoints of the Cardiopulmonary Meridian on the Autophagy in Rats with Acute Myocardial Ischemia.

Wushu acupoints are the five acupoints distributed below the human elbow and knee joint. They are all located on the same meridian and divided into five categories: Jing, Ying, Shu, Jing, and He. It has been shown that electroacupuncture (EA) at Shenmen point of heart meridian can improve acute myocardial ischemia (AMI) early. However, it is still unclear if all the Wushu acupoints of the heart meridian can improve AMI. Hence, this study emphasizes Wushu acupoints of heart meridian, compares them with Wushu acupoints of lung meridian, and studies the therapeutic effect of EA at Wushu acupoints on AMI and its possible mechanism. It also discusses the specificity of the heart meridian to heart disease. The AMI model is established by ligation of the left anterior descending coronary artery. The detection methods like the physiological recorder, TTC staining, ELISA, and so forth were used to determine the ECG, myocardial infarct size, serum myocardial enzymes, and myocardial tissue-related protein expression in rats. The heart rate (HR) and ST segment along with creatine kinase (CK), creatine kinase isoenzymes (CK-MB), lactate dehydrogenase (LDH), and myocardial infarctions increased after the induction with AMI. Furthermore, the expressions of PINK1 and Parkin protein also showed an increase. However, EA at Wushu acupoints in the heart meridian can reverse the above changes, whereas EA at the lung meridian exhibits limited effect. It is depicted that the heart meridian has a relatively specific relationship with the heart in a diseased state.

Effect of high-intensity interval training and coenzyme Q10 supplementation on cardiac apoptosis in obese male rats.

Obesity is associated with several metabolic disorders and myocardial apoptosis. The aim of this study was to determine the effect of high-intensity interval training (HIIT) and coenzyme Q10 (CoQ10) supplementation on myocardial apoptotic proteins in obese male rats. Forty-eight male Wistar rats were randomly assigned to 6 groups including non-obese control (NOC), baseline obese control (BOC), HIIT, CoQ10, HIIT + CoQ10, and control. NOC and BOC groups were sacrificed at the beginning of the study. Exercise groups were subjected to a HIIT program over 12 weeks. Rats in CoQ10 group were gavage-fed with 500 mg/kg-1 CoQ10 supplement throughout the study. The heart muscle was removed 48 hours after the last training session. The B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), cytochrome c, BH3-interacting domain death agonist (Bid), caspase-8, and caspase-3 protein expressions were analyzed using western blotting. Data were analyzed using independent t-test and two-way analysis of variance (ANOVA). The Bax, Bid, cytochrome c, caspase-8, caspase-3 proteins, and Bax/Bcl-2 were significantly higher in BOC group compared to NOC (P = 0.025, P = 0.0001, P = 0.013, P = 0.017, P = 0.010, P = 0.180, respectively). Moreover, Bcl-2 protein was lower in BOC compared to NOC group (P = 0.025). HIIT program decreased Bax/Bcl-2 ratio (P = 0.012), Bid (P = 0.0001), cytochrome c (P = 0.003), caspase-8 (P = 0.006), and caspase-3 (P = 0.002) proteins and increased Bcl-2 (P = 0.001) proteins in heart muscle. CoQ10 supplementation significantly increased Bcl-2 protein content (P = 0.001). HIIT exerts an anti-apoptotic effect in heart muscle of obese rats. Although the administration of CoQ10 increased Bcl-2 anti-apoptotic protein, it did not show a potent synergistic effect along with HIIT to reduce obesity-related myocardial apoptosis.

Peiminine inhibits myocardial injury and fibrosis after myocardial infarction in rats by regulating mitogen-activated protein kinase pathway.

Myocardial infarction promotes cardiac remodeling and myocardial fibrosis, thus leading to cardiac dysfunction or heart failure. Peiminine has been regarded as a traditional anti-fibrotic Chinese medicine in pulmonary fibrosis. However, the role of peiminine in myocardial infarction-induced myocardial injury and fibrosis remained elusive. Firstly, rat model of myocardial infarction was established using ligation of the left coronary artery, which were then intraperitoneally injected with 2 or 5 mg/kg peiminine once a day for 4 weeks. Echocardiography and haemodynamic evaluation results showed that peiminine treatment reduced left ventricular end-diastolic pressure, and enhanced maximum rate of increase/decrease of left ventricle pressure (± dP/dt max) and left ventricular systolic pressure, which ameliorate the cardiac function. Secondly, myocardial infarction-induced myocardial injury and infarct size were also attenuated by peiminine. Moreover, peiminine inhibited myocardial infarction-induced increase of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α production, as well as the myocardial cell apoptosis, in the rats. Thirdly, peiminine also decreased the myocardial fibrosis related protein expression including collagen I and collagen III. Lastly, peiminine reduced the expression of p38 and phosphorylation of extracellular signal-regulated kinase 1/2 in rat model of myocardial infarction. In conclusion, peiminine has a cardioprotective effect against myocardial infarction-induced myocardial injury and fibrosis, which can be attributed to the inactivation of mitogen-activated protein kinase pathway.

Electroacupuncture of acupoints of heart meridian ameliorates acute myocardial ischemia via Akt/mTOR pathway

To observe the effect of electroacupuncture (EA) on the expression of myocardial protein kinase B (Akt) and mammalian target of rapamycin (mTOR) in acute myocardial ischemia (AMI) rats. Thirty male SD rats were randomly divided into control, model and EA groups (n=10 in each group). The AMI model was established by occlusion of the descending anterior branch (DAB) of the left coronary artery. EA (2 Hz, 1-2 mA) was applied to bilateral "Shenmen" (HT7) and "Tongli" (HT5) for 20 min, once daily for consecutive 7 days. The electrocardiogram (ECG) of nape-xiphoid lead was recorded for assessing changes of myocardial ischemia. Histopathologic changes of the ischemic myocardial tissue were observed after H.E. staining and ultra-microstructural changes of cardiomyocytes observed by transmission electron microscopy (TEM). The expression levels of Akt, phosphorylated-Akt (p-Akt), mTOR and phosphorylated-mTOR (p-mTOR) in the myocardium were detected by Western blot, followed by calculating the ratios of p-Akt/Akt and p-mTOR/mTOR. Following ligature of DAB, the ECG-ST level was significantly increased in the model group in comparison with the control group (P<0.01). At 30 min after treatment, the ECG-ST level decreased significantly compared with the model group (P<0.01). At the end of the 7-day treatment period, the ECG-ST level increased compared with the model group (P<0.05). The levels of myocardial p-Akt and p-mTOR protein expression, and the ratios of p-Akt/Akt and p-mTOR/mTOR were significantly lower in the model group than those in the control group (P<0.01), and considerably increased in the EA group than in the model group (P<0.01). No significant differences were found among the three groups in the expression levels of Akt and mTOR proteins (P>0.05). Outcomes of H.E. staining and TEM showed damage of mitochondria and occurrence of a large number of autophagosomes in myocardiocytes in the model group, which was milder in the EA group. EA at HT5 and HT7 can improve AMI in AMI rats, which may be related to its effect in facilitating Akt/mTOR signaling.

Downregulation of Uncoupling Protein 2(UCP2) Mediated by MicroRNA-762 Confers Cardioprotection and Participates in the Regulation of Dynamic Mitochondrial Homeostasis of Dynamin Related Protein1 (DRP1) After Myocardial Infarction in Mice.

Acute myocardial infarction (MI) is one of the leading causes of death in the world, and its pathophysiological mechanisms have not been fully elucidated. The purpose of this study was to investigate the role and mechanism of uncoupling protein 2 (UCP2) after MI in mouse heart. Here, we examined the expression and role of UCP2 in mouse heart 4 weeks after MI. The expression of UCP2 was detected by RT-PCR and western blotting. Cardiac function, myocardial fibrosis, and cardiomyocyte apoptosis were assessed by echocardiography and immunohistochemistry. Phosphatase dynamin-related protein1 (P-DRP1) and myocardial fibrosis-related proteins were measured. Cardiomyocytes were exposed to hypoxia for 6 h to mimic the model of MI. Mdivi, an inhibitor of P-DRP1, was used to inhibit DRP1-dependent mitochondrial fission. Mitochondrial superoxide, membrane potential, oxygen consumption rate, and cardiomyocyte apoptosis were detected after hypoxia. It is shown mitochondrial superoxide, membrane potential, oxygen consumption rate, and cardiomyocyte apoptosis were dependent on the level of P-DRP1. UCP2 overexpression reduced cardiomyocyte apoptosis (fibrosis), improved cardiac function and inhibit the phosphorylation of DRP1 and the ratio of P-DRP1/DRP1. However, inhibition of DRP1 by mdivi did not further reduce cell apoptosis rate and cardiac function in UCP2 overexpression group. In addition, bioinformatics analysis, luciferase activity, and western blot assay proved UCP2 was a direct target gene of microRNA-762, a up-regulated microRNA after MI. In conclusion, UCP2 plays a protective role after MI and the mechanism is involved in microRNA-762 upstream and DRP1-dependent mitochondrial fission downstream.

Abstract P1-08-05: Expression of the human APOE4 genotype modulates Doxorubicin cardiotoxicity in mice

Abstract Background: The human apolipoprotein E (APOE) gene has three major allelic variants: APOE2, -E3 and -E4. Carriers of the E4 allele are at increased risk for Alzheimer’s and other age-related neurodegenerative disorders. Recently, APOE4 has been associated with risk for cancer-related cognitive decline following chemotherapy with Doxorubicin (DOX), a frequent component of adjuvant therapy for breast cancer. To date, the mechanisms for APOE4 neurotoxicity are unknown, but may greater vulnerability to oxidative stress. DOX also causes dose-dependent cardiotoxicity, but a role for APOE4 in heart damage has not been previously demonstrated. Objective: To determine whether APOE4 predisposes to DOX-induced damage to the myocardium. Methods: C57Bl/6 mice (5-7 mo and 14-18 mo, male and female) with human APOE3 or APOE4 homozygous knock-in (=hAPOE3 and hAPOE4, respectively) were subjected to a single IP injection of saline (Control) or DOX (10 mg/kg, or 5 weekly injections of 5 mg/kg, =DOX), and monitored between 3-45 days afterward. Left ventricular systolic function was quantitated using echocardiography (Vevo 3100) at baseline, during and at the end of each study. TUNEL assay was used to identify apoptotic cells. Collagen was imaged using Masson’s Trichrome staining. Myocardial protein and RNA were extracted from the left ventricle and was analyzed by immunoblotting, realtime PCR and RNASeq on an Illumina NexGen platform.Results: Aged female APOE4 mice had more myocardial collagen at baseline (8.93±1.23 vs. 6.06±1.44%). Baseline cardiac function were similar at baseline in the 2 mouse strains. Three days after DOX, apoptotic myocytes were more abundant in hAPOE4 vs hAPOE3 hearts (1.2 vs. 4.6 TUNEL+ cells per section, n= 5, p&amp;lt;0.05). After DOX treatment, hAPOE4 mice had greater declines in left ventricular ejection fraction (EF, 46.9 vs 62.1%, p = hAPOE4 vs hAPOE3, p &amp;lt; 0.001). Quantitative PCR, RNASeq and protein studies revealed multiple differences in the cardiac transcriptome between hAPOE3 and hAPOE4 mice both at baseline and after DOX treatment. Baseline expression of MEF2D, a key driver of the myocardial stress response, was increased in hAPOE4 vs APOE3, driven by a 3500x (p = 4.76E-17) in transcript variant 1. Robust differences were seen in expression of c-Jun, JunB, and myosin heavy chain genes Myh6 and Myh7 at baseline and following DOX treatment at day 3. Growth Arrest And DNA Damage Inducible Alpha(GADD45A) gene expression was ~2x higher in hAPOE4 mice at baseline, but was similar to hAPOE3 mice by day 14 after DOX. Protein levels of caspase-1, an essential regulator of inflammation and the innate immune response, increased with age in hAPOE3 mice. In contrast, levels of casp-1 were lower in hAPOE4 and did not increase with age (p= 0.002 for APOE4 vs APOE3 at 17 mo). Conclusions: These findings support a model in which the APOE4 allele increases vulnerability to DOX-induced cardiac damage through mechanisms that involve significant and complex alterations in the response to oxidative stress and/or damage signaling. Studies are ongoing to further elucidate transcriptional differences conferred by APOE4, to analyze substrates downstream of caspase-1 affected by APOE4, and to determine the extent to which APOE4 may contribute to DOX toxicity in patient populations. Citation Format: Guannan Wang, G. William Rebeck, Olga Rodriguez, Marc E. Lippman, Nanette H. Bishopric. Expression of the human APOE4 genotype modulates Doxorubicin cardiotoxicity in mice [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-05.

Expression of Iron Metabolism Proteins in Patients with Chronic Heart Failure.

In heart failure, iron deficiency is a common comorbid disease that negatively influences exercise tolerance, number of hospitalizations and mortality rate, and this is why iron iv supplementation is recommended. Little is known about the changes in iron-related proteins in the human HF myocardium. The purpose of this study was to assess iron-related proteins in non-failing (NFH) vs. failing (FH) human myocardium. The study group consisted of 58 explanted FHs; control consisted of 31 NFHs unsuitable for transplantation. Myocardial proteins expressions: divalent metal transporter (DMT-1); L-type calcium channel (L-CH); transferrin receptors (TfR-1/TfR-2); ferritins: heavy (FT-H) or light (FT-L) chain, mitochondrial (FT-MT); ferroportin (FPN), regulatory factors and oxidative stress marker: 4-hydroxynonenal (4-HNE). In FH, the expression in almost all proteins responsible for iron transport: DMT-1, TfR-1, L-CH, except TfR-2, and storage: FT-H/-L/-MT were reduced, with no changes in FPN. Moreover, 4-HNE expression (pg/mg; NFH 10.6 ± 8.4 vs. FH 55.7 ± 33.7; p < 0.0001) in FH was increased. HNE-4 significantly correlated with DMT-1 (r = −0.377, p = 0.036), L-CH (r = −0.571, p = 0.001), FT-H (r = −0.379, p = 0.036), also FPN (r = 0.422, p = 0.018). Reducing iron-gathering proteins and elevated oxidative stress in failing hearts is very unfavorable for myocardiocytes. It should be taken into consideration before treatment with drugs or supplements that elevate free oxygen radicals in the heart.

Activation of PKC results in improved contractile effects and Ca2+ cycling by inhibition of PP2A-B56α.

Protein phosphatase 2A (PP2A) represents a heterotrimer that is responsible for the dephosphorylation of important regulatory myocardial proteins. This study was aimed to test whether the phosphorylation of PP2A-B56α at Ser41 by PKC is involved in the regulation of myocyte Ca2+ cycling and contraction. For this purpose, heart preparations of wild-type (WT) and transgenic mice overexpressing the nonphosphorylatable S41A mutant form (TG) were stimulated by administration of the direct PKC activator phorbol 12-myristate 13-acetate (PMA), and functional effects were studied. PKC activation was accompanied by the inhibition of PP2A activity in WT cardiomyocytes, whereas this effect was absent in TG. Consistently, the increase in the sarcomere length shortening and the peak amplitude of Ca2+ transients after PMA administration in WT cardiomyocytes was attenuated in TG. However, the costimulation with 1 µM isoprenaline was able to offset these functional deficits. Moreover, TG hearts did not show an increase in the phosphorylation of the myosin-binding protein C after administration of PMA but was detected in corresponding WT. PMA modulated voltage-dependent activation of the L-type Ca2+ channel (LTCC) differently in the two genotypes, shifting V1/2a by +1.5 mV in TG and by -2.4 mV in WT. In the presence of PMA, ICaL inactivation remained unchanged in TG, whereas it was slower in corresponding WT. Our data suggest that PKC-activated enhancement of myocyte contraction and intracellular Ca2+ signaling is mediated by phosphorylation of B56α at Ser41, leading to a decrease in PP2A activity.NEW & NOTEWORTHY The importance of the serine-41 phosphorylation site on B56α in reducing PP2A activity was demonstrated for the first time using a transgenic mutation model. Direct activation of PKC inhibits PP2A, leading to increased phosphorylation of MyBP-C in cardiomyocytes. The increased phosphorylation of contractile proteins is influenced by the PKC-phosphoB56α-PP2A signaling cascade resulting in improved intracellular Ca2+ handling and enhanced contractility and relaxation. PKC-mediated inhibition of PP2A also leads to modulation of the LTCC activation and inactivation kinetics.

Myocardial Proteomics Based on Smart Fog Computing and Its Application in Sports.

How to strengthen physical fitness to improve the effect and efficiency of sports is an important research direction worthy of research. In response to these problems and limitations, Smart Fog Computing technology is introduced in this paper. Taking rats as the research object, the effective quantitative analysis and research of aerobic exercise on myocardial proteome are achieved through combining the business scope of myocardial proteomics, and connecting corresponding continuous aerobic exercises, verified by simulation analysis. The simulation research results show that the smart fog calculation is effective. For moderate-intensity aerobic exercise, the expression and intensity of the corresponding myocardial protein are changed significantly, and the corresponding heart becomes larger; meanwhile, moderate aerobic exercise can improve the metabolism and enhance digestive ability.

Cardiac myosin mechanochemistry underlies left ventricular dysfunction in human ischemic heart failure

Prolonged occlusion of the coronary arteries can lead to ischemic heart failure (IHF). Molecular changes within the myocardium during IHF are not fully understood. Accordingly, we aimed to characterize the biophysical properties of myocardial contractile proteins in IHF. Ten age-matched human males with similar BMI were included in the study. Cardiac biopsies of left ventricular free wall were taken from patients with ischemic heart failure (IHF, n=5) at the time of heart transplant or from organ donors with no previous history of ischemic heart failure (controls, n=5).

Genetic Variants Associated With Sudden Cardiac Death in Victims With Single Vessel Coronary Artery Disease and Left Ventricular Hypertrophy With or Without Fibrosis

Objective: Cardiac hypertrophy with varying degrees of myocardial fibrosis is commonly associated with coronary artery disease (CAD) related sudden cardiac death (SCD), especially in young victims among whom patterns of coronary artery lesions do not entirely appear to explain the cause of SCD. Our aim was to study the genetic background of hypertrophy, with or without fibrosis, among ischemic SCD victims with single vessel CAD.Methods: The study population was derived from the Fingesture study, consisting of all autopsy-verified SCDs in Northern Finland between the years 1998 and 2017 (n = 5,869). We carried out targeted next-generation sequencing using a panel of 174 genes associated with myocardial structure and ion channel function in 95 ischemic-SCD victims (mean age 63.6 ± 10.3 years; 88.4% males) with single-vessel CAD in the absence of previously diagnosed CAD and cardiac hypertrophy with or without myocardial fibrosis at autopsy.Results: A total of 42 rare variants were detected in 43 subjects (45.3% of the study subjects). Five variants in eight subjects (8.4%) were classified as pathogenic or likely pathogenic. We observed 37 variants of uncertain significance in 39 subjects (40.6%). Variants were detected in myocardial structure protein coding genes, associated with arrhythmogenic right ventricular, dilated, hypertrophic and left ventricular non-compaction cardiomyopathies. Also, variants were detected in ryanodine receptor 2 (RYR2), a gene associated with both cardiomyopathies and catecholaminergic polymorphic ventricular tachycardias.Conclusions: Rare variants associated with cardiomyopathies, in the absence of anatomic evidence of the specific inherited cardiomyopathies, were common findings among CAD-related SCD victims with single vessel disease and myocardial hypertrophy found at autopsies, suggesting that these variants may modulate the risk for fatal arrhythmias and SCD in ischemic disease.

Sodium-Glucose Co-transporter-2 Inhibitor of Dapagliflozin Attenuates Myocardial Ischemia/Reperfusion Injury by Limiting NLRP3 Inflammasome Activation and Modulating Autophagy.

Background: The sodium-glucose co-transporter-2 (SGLT-2) inhibitor dapagliflozin improves cardiovascular outcomes in patients with type 2 diabetes in a manner that is partially independent of its hypoglycemic effect. These observations suggest that it may exert a cardioprotective effect by another mechanism. This study explored the effects of dapagliflozin on myocardial ischemia/reperfusion injury in a mouse model.Materials and Methods: For the in vivo I/R studies, mice received 40 mg/kg/d dapagliflozin, starting 7 days before I/R. Evans Blue/TTC double-staining was used to determine the infarct size. Serum levels of cTnI, CK-MB, and LDH were measured. Inflammation, autophagy protein expression, and caspase-1 activity changes were measured at the protein level. Primary cardiomyocytes were used to investigate the direct effect of dapagliflozin on cardiomyocytes and to verify whether they have the same effect as observed in in vivo experiments.Result: A high dose of dapagliflozin significantly reduced infarct size and decreased the serum levels of cTnI, CK-MB, and LDH. Dapagliflozin also reduced serum levels of IL-1β, reduced expression of myocardial inflammation-related proteins, and inhibited cardiac caspase-1 activity. The treatment restored autophagy flux and promoted the degradation of autophagosomes. Relief of inflammation relied on autophagosome phagocytosis of NLRP3 and autophagosome clearance after lysosome improvement. 10 μM dapagliflozin reduced intracellular Ca2+ and Na+ in primary cardiomyocytes, and increasing NHE1 and NCX expression mitigated dapagliflozin effects on autophagy.Conclusion: Dapagliflozin protects against myocardial ischemia/reperfusion injury independently of its hypoglycemic effect. High-dose dapagliflozin pretreatment might limit NLRP3 inflammasome activation and mediate its selective autophagy. Dapagliflozin directly acts on cardiomyocytes through NHE1/NCX.