Myocyte Cell Death Research Articles

Enhancing Sarcolemmal Membrane Repair Using a Novel Protein Therapeutic Approach can Improve Membrane Integrity in Duchenne Muscular Dystrophy Cells and Mouse Models

Duchenne Muscular Dystrophy (DMD) is a fatal neuromuscular disease involving progressive cardiac and skeletal myocyte cell death and subsequent muscle degeneration. DMD and Becker muscular dystrophy (BMD) result from mutations in the dystrophin gene that compromise the structural integrity of the sarcolemma. While there has been important recent progress in the treatment of DMD, the currently available therapeutics have significant limitations. Thus, novel therapies that address the compromised sarcolemmal membrane integrity are an unmet medical need. Tripartite motif protein 72, or mitsugumin 53 (TRIM72/MG53), facilitates the sarcolemma repair response after disruption of the membrane. Loss of TRIM72/MG53 function in mice leads to a progressive myopathy. Exogenous delivery of recombinant human MG53 protein (rhMG53) can increase sarcolemmal membrane repair in many different cell types and ameliorate pathology in models of DMD and multiple limb girdle muscular dystrophies. Despite the promise of rhMG53 as a therapeutic invention the development of this protein to treat DMD has been complicated by findings showing that high levels of the protein correlate with metabolic dysfunction in animal models and some human patients. Deletion analysis of the major domains of the rhMG53 protein allowed us to design an improved version of rhMG53 called MyoTRIM. MyoTRIM contains amino acid replacements to eliminate metabolic effects and improve solubility while maintaining membrane repair effects. One set of modifications inactivate the E3 ligase activity of the protein, which revealed this function is dispensable for subcellular localization of the protein. TRIM72/MG53 is reported to mediate this effect by binding phosphatidylserine (PS) at membrane injury sites, so we demonstrate that MyoTRIM can bind PS-coated beads to illustrate that carboxy-terminal domains are required for effcient PS binding rather than the E3 ligase function. External application of MyoTRIM can increase membrane repair capacity in a variety of cell-based assays using DMD and BMD patient myoblasts and myotubes. We also establish that MyoTRIM can increase membrane repair and prevent eccentric contraction induced injury in a dystrophin deficient mouse model. Taken together, these results provide mechanistic insight into rhMG53 function and indicate that MyoTRIM can recapitulate the therapeutic effects on sarcolemmal membrane repair while eliminating E3 ligase activity and associated side effects. This work was supported by the Offce of the Assistant Secretary of Defense for Health Affairs through the Duchenne Muscular Dystrophy Research Program (DMDRP) under Translational Research Award HT9425-23-1-0940. The opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Angiotensin II Is Involved in MLKL Activation During the Development of Heart Failure Following Myocardial Infarction in Rats.

Several reports assume that myocardial necroptotic cell death is induced during the development of chronic heart failure. Although it is well accepted that angiotensin II induces apoptotic cell death of cardiac myocytes, the involvement of angiotensin II in the induction of myocardial necroptosis during the development of heart failure is still unknown. Therefore, we examined the role of angiotensin II in myocardial necroptosis using rat failing hearts following myocardial infarction and cultured cardiomyocytes. We found that administration of azilsartan, an angiotensin II AT1 receptor blocker, or trandolapril, an angiotensin-converting enzyme inhibitor, to rats from the 2nd to the 8th week after myocardial infarction resulted in preservation of cardiac function and attenuation of mixed lineage kinase domain-like (MLKL) activation. Furthermore, the ratio of necroptotic cell death was increased in neonatal rat ventricular cardiomyocytes cultured with conditioned medium from rat cardiac fibroblasts in the presence of angiotensin II. This increase in necroptotic cells was attenuated by pretreatment with azilsartan. Furthermore, activated MLKL was increased in cardiomyocytes cultured in conditioned medium. Pretreatment with azilsartan also prevented the conditioned medium-induced increase in activated MLKL. These results suggest that angiotensin II contributes to the induction of myocardial necroptosis during the development of heart failure.

SMYD1a protects the heart from ischemic injury by regulating OPA1-mediated cristae remodeling and supercomplex formation

SMYD1, a striated muscle-specific lysine methyltransferase, was originally shown to play a key role in embryonic cardiac development but more recently we demonstrated that loss of Smyd1 in the murine adult heart leads to cardiac hypertrophy and failure. However, the effects of SMYD1 overexpression in the heart and its molecular function in the cardiomyocyte in response to ischemic stress are unknown. In this study, we show that inducible, cardiomyocyte-specific overexpression of SMYD1a in mice protects the heart from ischemic injury as seen by a > 50% reduction in infarct size and decreased myocyte cell death. We also demonstrate that attenuated pathological remodeling is a result of enhanced mitochondrial respiration efficiency, which is driven by increased mitochondrial cristae formation and stabilization of respiratory chain supercomplexes within the cristae. These morphological changes occur concomitant with increased OPA1 expression, a known driver of cristae morphology and supercomplex formation. Together, these analyses identify OPA1 as a novel downstream target of SMYD1a whereby cardiomyocytes upregulate energy efficiency to dynamically adapt to the energy demands of the cell. In addition, these findings highlight a new epigenetic mechanism by which SMYD1a regulates mitochondrial energetics and functions to protect the heart from ischemic injury.

Mitochondrial GRK2 is a Novel Regulator of Cardiac Energetics

G protein‐coupled receptor (GPCR) kinase 2 (GRK2) is highly expressed in the heart, where during injury or heart failure (HF), both its levels and activity increase. GRK2 is canonically studied in the context of GPCR phosphorylation; however, noncanonical activities of GRK2 have emerged and it is now appreciated that GRK2 has a large non‐GPCR interactome. For example, in cardiac myocytes, GRK2 translocates from the cytosol to mitochondria (mtGRK2) following oxidative stress or ischemia injury, and this pool of mtGRK2 is associated with negative effects on metabolism and also induces myocyte cell death. However, the mechanisms by which mtGRK2 contributes to cardiac dysfunction and HF are not fully understood. We hypothesized that mtGRK2 could have novel substrates and phosphorylate proteins involved in mitochondrial bioenergetics, thus contributing to our previously established post‐injury phenotype. Stress‐induced mitochondrial translocation of cytosolic GRK2 was validated in cell and animal models and the mtGRK2 interactome was identified using liquid chromatography‐mass spectroscopy (LCMS). Proteomics analysis identified mtGRK2 interacting proteins which were involved in mitochondrial dysfunction, bioenergetics, and OXPHOS, particularly complexes I, II, IV and V of the electron transport chain (ETC). Specifically, mtGRK2 interactions with Complex V (ATP synthase) subunits were particularly increased following stress. We established that mtGRK2 phosphorylates ATP synthase on the F1 catalytic barrel, which is critical for oxidative phosphorylation and ATP production. We have also determined that alterations in either the levels or activity of GRK2 appear to alter ATP synthase enzymatic activity in vitro. Excitingly, in vivo data suggest that reducing levels of GRK2 in a mouse model of myocardial infarction prevents the post‐injury reduction in ATP synthesis. We are currently assessing the ability of the SSRI drug paroxetine, a GRK2 inhibitor, to preserve mitochondrial bioenergetics in a transgenic GRK2 mouse model. Thus, phosphorylation of the ATP synthesis machinery by mtGRK2 may contribute to the impaired mitochondrial phenotype observed in injured or failing heartssuch as reduced fatty acid metabolism and substrate utilization. These data uncover a druggable, novel target for rescuing cardiac function in patients with injured and/or failing hearts.

249 Prognostic role of diagnostic criteria of acute myocardial infarction

Abstract Aims The term acute myocardial infarction (AMI) reflects cell death of cardiac myocytes caused by ischaemia. The Fourth Universal Definition of Myocardial Infarction (UDMI) defined AMI by a typical rise and fall in the level of biochemical markers of myocardial necrosis together with criteria of myocardial ischaemia. However, the prognostic role of each single diagnostic criteria has never been explored. To evaluate the prognostic role of the different diagnostic criteria of AMI according to the Fourth UDMI. Methods and results We enrolled all consecutive patients with AMI admitted from 2016 to 2019. We used a combination of criteria, according to the current ESC guidelines, to meet the diagnosis, namely the detection of an increase and/or decrease of high-sensitivity cardiac troponin I, with at least one value above the 99th percentile of the upper reference limit and at least one of the following: symptoms of ischaemia; ECG changes (new ST-T changes or new left bundle branch block); development of pathological Q waves in the ECG; imaging evidence of new loss of viable myocardium or new regional wall motion abnormality, in our study evaluated by transthoracic echocardiogram. All-cause mortality and a composite endpoint of all-cause mortality, re-hospitalization for heart failure, and myocardial re-infarction were collected. The predictive value of diagnostic criteria alone and its association were evaluated using Kaplan–Meier survival curves and subsequent Cox-regression analysis to find independent predictors of adverse events. 2386 patients were evaluated. The median follow-up time was 23.3 ± 14.5 months. The total number of events was 703 (29.5%). Kaplan–Meier curves showed that major adverse cardiac events (MACEs) were statistically different depending on the diagnostic criteria of AMI at admission. Particularly, clinical criteria alone showed a better predictive value (P < 0.001) than other diagnostic AMI criteria. Multivariable Cox-regression model demonstrated that clinical criteria were the independent predictor of good prognosis in patients with AMI (HR = 0.43; 95% CI: 0.28–0.67; P < 0.001). Conversely, the other diagnostic criteria (electrocardiographic and echocardiographic) and the combination of all diagnostic criteria were not independent prognostic factors of MACEs (HR = 1.1; 95% CI: 0.6–2.4, P = 0.6; HR = 1.1; 95% CI: 0.7–1.9, P = 0.6; HR = 0.9; 95% CI: 0.7–1.0, P = 0.2, respectively). Conclusions Our data suggest that the prognosis is considerably better among patients with a diagnosis of AMI if clinical criteria alone are present at admission. We also demonstrated that clinical criteria are a strong prognostic predictor of good outcomes in patients with AMI. We hypothesize that the absence of electrocardiographic and echocardiographic alterations could indirectly indicate a smaller infarct sizes that contribute to patients’ outcome.

Prognostic role of diagnostic criteria of acute myocardial infarction

Abstract Background The term acute myocardial infarction (AMI) reflects cell death of cardiac myocytes caused by ischaemia. The Fourth Universal Definition of Myocardial Infarction (UDMI) defined AMI by a typical rise and fall in the level of biochemical markers of myocardial necrosis together with criteria of myocardial ischaemia. However, the prognostic role of each single diagnostic criteria has never been explored. Purpose To evaluate the prognostic role of the different diagnostic criteria of AMI according to the Fourth UDMI. Methods We enrolled all consecutive patients with AMI admitted from 2016 to 2019. We used a combination of criteria, according to the current ESC guidelines, to meet the diagnosis, namely the detection of an increase and/or decrease of high-sensitivity cardiac troponin I, with at least one value above the 99th percentile of the upper reference limit and at least one of the following: symptoms of ischaemia; ECG changes (new ST-T changes or new LBBB); development of pathological Q waves in the ECG; imaging evidence of new loss of viable myocardium or new regional wall motion abnormality, in our study evaluated by transthoracic echocardiogram. All-cause mortality and a composite endpoint of all-cause mortality, re-hospitalization for heart failure and myocardial re-infarction were collected. The predictive value of diagnostic criteria alone and its association were evaluated using Kaplan-Meier survival curves and subsequent Cox-regression analysis to find independent predictors of adverse events. Results 2386 patients were evaluated. The median follow-up time was 23.3±14.5 months. The total number of events was 703 (29.3%). Kaplan-Meier curves showed that major adverse cardiac events (MACE) were statistically different depending on the diagnostic criteria of AMI at admission. Particularly, clinical criteria alone showed a better predictive value (p<0.001) than other diagnostic AMI criteria. Multivariable Cox-regression model demonstrated that clinical criteria was the independent predictor of good prognosis in patients with AMI (HR=0.43; CI 95% 0.28–0.67; p<0.001). Conversely, the others diagnostic criteria (electrocardiographic and echocardiographic) and the combination of all diagnostic criteria were not independent prognostic factors of MACE (HR=1.1 CI 95% 0.6–2.4, p=0.6; HR=1.1 CI 95% 0.7–1.2, p=0.6; HR=0.9 CI 95% 0.7–1.0, p=0.8 respectively). Conclusions Our data suggest that the prognosis is considerably better among patients with a diagnosis of AMI if clinical criteria alone are present at admission. We also demonstrated that clinical criteria are a strong prognostic predictor of good outcomes in patients with AMI. We hypothesize that the absence of electrocardiographic and echocardiographic alterations could indirectly indicate a smaller infarct sizes that contribute to patients' outcome. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): None

"Regulatory Roles for miR-126 in Inflammatory Response and Programmed Death of Cardiac Myocytes Driven by Acidosis During Simulated Ischemia"

Cardiac myocyte cell death by apoptosis accompanies heart disease of both ischemic and nonischemic origin...

Molecular mechanisms of anthracycline cardiovascular toxicity.

Anthracyclines are effective chemotherapeutic agents, commonly used in the treatment of a variety of hematologic malignancies and solid tumors. However, their use is associated with a significant risk of cardiovascular toxicities and may result in cardiomyopathy and heart failure. Cardiomyocyte toxicity occurs via multiple molecular mechanisms, including topoisomerase II-mediated DNA double-strand breaks and reactive oxygen species (ROS) formation via effects on the mitochondrial electron transport chain, NADPH oxidases (NOXs), and nitric oxide synthases (NOSs). Excess ROS may cause mitochondrial dysfunction, endoplasmic reticulum stress, calcium release, and DNA damage, which may result in cardiomyocyte dysfunction or cell death. These pathophysiologic mechanisms cause tissue-level manifestations, including characteristic histopathologic changes (myocyte vacuolization, myofibrillar loss, and cell death), atrophy and fibrosis, and organ-level manifestations including cardiac contractile dysfunction and vascular dysfunction. In addition, these mechanisms are relevant to current and emerging strategies to diagnose, prevent, and treat anthracycline-induced cardiomyopathy. This review details the established and emerging data regarding the molecular mechanisms of anthracycline-induced cardiovascular toxicity.

Exercise triggers CAPN1-mediated AIF truncation, inducing myocyte cell death in arrhythmogenic cardiomyopathy.

Myocyte death occurs in many inherited and acquired cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), a genetic heart disease plagued by the prevalence of sudden cardiac death. Individuals with ACM and harboring pathogenic desmosomal variants, such as desmoglein-2 (DSG2), often show myocyte necrosis with progression to exercise-associated heart failure. Here, we showed that homozygous Dsg2 mutant mice (Dsg2 mut/mut), a model of ACM, die prematurely during swimming and display myocardial dysfunction and necrosis. We detected calcium (Ca2+) overload in Dsg2 mut/mut hearts, which induced calpain-1 (CAPN1) activation, association of CAPN1 with mitochondria, and CAPN1-induced cleavage of mitochondrial-bound apoptosis-inducing factor (AIF). Cleaved AIF translocated to the myocyte nucleus triggering large-scale DNA fragmentation and cell death, an effect potentiated by mitochondrial-driven AIF oxidation. Posttranslational oxidation of AIF cysteine residues was due, in part, to a depleted mitochondrial thioredoxin-2 redox system. Hearts from exercised Dsg2 mut/mut mice were depleted of calpastatin (CAST), an endogenous CAPN1 inhibitor, and overexpressing CAST in myocytes protected against Ca2+ overload-induced necrosis. When cardiomyocytes differentiated from Dsg2 mut/mut embryonic stem cells (ES-CMs) were challenged with β-adrenergic stimulation, CAPN1 inhibition attenuated CAPN1-induced AIF truncation. In addition, pretreatment of Dsg2 mut/mut ES-CMs with an AIF-mimetic peptide, mirroring the cyclophilin-A (PPIA) binding site of AIF, blocked PPIA-mediated AIF-nuclear translocation, and reduced both apoptosis and necrosis. Thus, preventing CAPN1-induced AIF-truncation or barring binding of AIF to the nuclear chaperone, PPIA, may avert myocyte death and, ultimately, disease progression to heart failure in ACM and likely other forms of cardiomyopathies.

Abstract 15737: Deficiency of Mitochondrial Calcium Uniporter Protects Mouse Hearts From Iron Overload by Attenuating Ferroptosis

Iron (Fe) plays essential roles in many physiological processes. Hereditary hemochromatosis and frequent blood transfusions result in iron overload (IO) and dysfunction of iron-deposited organs including the heart. Although IO-induced cardiomyopathy remains a significant clinical challenge, the underlying mechanism is not well defined. In the present study, we aim to assess the involvement of the mitochondrial Ca uniporter (mCU) in IO-induced cardiac contractile dysfunction and ferroptosis. In a chronic IO model, after receiving Fe (dextran, i.p. 0.6 mg/g, 3 days/wk) for 6 weeks, systolic function (LVEF and LVFS) was reduced in mCU +/+ (WT) compared to mCU -/- (mCU KO). This observation was confirmed in isolated ventricular myocytes where we similarly detected a significant decrease in cell shortening in WT, but not mCU KO myocytes. We found lower Fe levels in mitochondria from mCU KO myocytes compared to WT, while observing the same level of Fe deposition in heart tissue from both groups. The mitochondrial ROS level was lower in mCU KO myocytes vs. WT. Long term cardiac dysfunction may result in myocyte cell death, however, we did not detect apoptosis (TUNEL) in either mCU KO or in WT hearts with cardiac dysfunction after chronic IO. The lipid oxidation level was increased with Fe load suggesting ferroptosis may be involved in IO-induced cardiomyopathy, which was supported by the observation that administration of the selective ferroptosis inhibitor ferrostatin-1 reduced lipid oxidation (4-HNE) and maintained heart function. To further determine the role of mCU in IO-mediated ferroptosis in the heart, we used isolated myocytes from WT and mCU KO and conducted Live/Dead cell viability assays. While Fe (FAC, 0.1-5 mM) induced ferroptosis in a dose-dependent manner, it was prevented by ferrostatin-1 (10 μM), the Fe chelator 2,2'-bipyridyl (2 mM), and MitoTEMPO (5 μM), respectively. Fe-induced ferroptosis in mCU KO myocytes did not occur while the lipid oxidation level (Liperfluo) remained low. In conclusion, mitochondrial Fe uptake, presumably mediated by mCU, accounts for the molecular/cellular mechanisms for Fe-induced myocyte ferroptosis and cardiomyopathy. Cardiac-specific deficiency of mCU prevents the development of Fe induced cardiomyopathy.

BNIP3 in Lung Cancer: To Kill or Rescue?

Simple SummaryBcl-2/adenovirus E1B 19kDa interacting protein 3 (BNIP3) is a pro-apoptotic BH3-only protein of the Bcl-2 family. Its function in various biological processes was described. Although potential involvement of BNIP3 in cancer progression has been discussed in many review articles, its specific role in lung cancer is still unclear. In this review, we shed light on the BNIP3‘s role in different types of cancer in general and lung cancer, in particular, as well as suggested its potential for targeting therapy of lung cancer.Bcl-2/adenovirus E1B 19kDa interacting protein 3 (BNIP3) is a pro-apoptotic BH3-only protein of the Bcl-2 family. Initially, BNIP3 was described as one of the mediators of hypoxia-induced apoptotic cell death in cardiac myocytes and neurons. Besides apoptosis, BNIP3 plays a crucial role in autophagy, metabolic pathways, and metastasis-related processes in different tumor types. Lung cancer is one of the most aggressive types of cancer, which is often diagnosed at an advanced stage. Therefore, there is still urgent demand for reliable biochemical markers for lung cancer and its efficient treatment. Mitochondria functioning and mitochondrial proteins, including BNIP3, have a strong impact on lung cancer development and progression. Here, we summarized current knowledge about the BNIP3 gene and protein features and their role in cancer progression, especially in lung cancer in order to develop new therapeutic approaches associated with BNIP3.

ARVC specific autoantibody identifies cardiac sarcoidosis and correlates with inflammation activity

Abstract Introduction Cardiac sarcoidosis (CS) is an inflammatory granulomatous disease of unknown origin. CS and arrhythmogenic right ventricular cardiomyopathy (ARVC) are overlapping syndromes. With both, patients are at increased risk of ventricular arrhythmias and sudden cardiac death. However, the diagnosis of CS is challenging, especially in patients with no extracardiac involvement, but correct diagnosis has large therapeutic impact. Recently, a novel diagnostic autoantibody (anti-DSG2 Ab) was identified in ARVC. We sought to identify this antibody in CS patients and correlate its levels with inflammation activity using cardiac positron-emission-tomography (18-FDG-PET). Methods Recombinant human desmoglein-2 (DSG2) proteins on western blots were exposed to sera as well as purified IgG of 14 patients with sarcoidosis (all confirmed by histology) and 6 controls (1 ARVC patient (positive control) and 5 healthy control subjects (negative control)). Clinical patient characteristics were correlated to detected antibody intensity levels. Results The sarcoidosis cohort comprised 43% (6/14) male patients and the average age was 50±12 years. Anti-DSG2 Abs were identified in 43% (6/14) and were detected faintly (below cut off level) in 21% (3/14) of all sarcoidosis patients. Antibody was also present in the ARVC patient (1/1) and was absent in all control subjects (5/5). Myocardial inflammation was present in 18-FDG PET imaging in all CS patients with positive anti-DSG2 Abs, corresponding to an average SUV (standardized uptake value) of 8.1±4.2. In patients with faint or no antibody, the SUV values were significantly lower with 1.2±2.1 and 3.2±4.0, respectively (P=0.044, one-way ANOVA). The Pearson correlation coefficient (R) was 0.6 (P=0.037) for SUV vs. higher antibody levels assessed by pixel count of the western blot bands for purified IgG. Conclusions Anti-DSG2 Abs are not only a specific biomarker for ARVC, but are also found in CS, suggesting a similar pathophysiological mechanism in these overlapping syndromes, both involving cardiac inflammation and myocyte cell death. Moreover, antibody levels correlate with disease activity on cardiac PET imaging. Larger cohorts are necessary to confirm these findings. Funding Acknowledgement Type of funding source: None

Doxorubicin-induces NFAT/Fas/FasL cardiac apoptosis in rats through activation of calcineurin and P38 MAPK and inhibition of mTOR signalling pathways.

This study investigated the role of NFAT/Fas/FasL axis in cardiomyocyte apoptosis following doxorubicin (DOX) treatment in rats and evaluated the involvement and regulation of all NFAT members in cardiac apoptosis. Forty adult male Wistar rats were divided equally into control or DOX-treated groups (15mg/kg over 2weeks). Cardiomyocytes were cultured and pre-incubated with various inhibitors and activators (10μmol/L) prior to DOX exposure (1μmol/L). In the left ventricles and cultured cells, DOX increased cytoplasmic protein levels of cytochrome C, Bax and increased the activities of caspase-8, caspase3, ERK1/2, JNK, and P38 mitogen-activated protein kinases (MAPKs), reducing levels of Bcl-2 and the activity of mTOR, and inducing cell death. In addition, DOX enhanced mRNA and protein levels of Fas and FasL. Furthermore, the nuclear and cytoplasmic levels of NFAT1 and nuclear accumulation of NFAT2-4were increased with DOX treatment. The inhibition of calcineurin with FK506 significantly inhibited the nuclear levels of NFAT2 and NFAT4 and the inhibition of P38 MAPK with SB203580 inhibited the nuclear and cytoplasmic accumulation of NFAT1. However, the activation of mTOR by IGF-1 significantly lowered NFAT3. In conclusion, NFAT/Fas/FasL-induced cell death in cardiac myocytes of DOX-treated rats is regulated, at least, by the activation of calcineurin and P38 MAPK and inhibition of mTOR.

Cell Killing by Reovirus: Mechanisms and Consequences.

Infection of host cells by mammalian reovirus in culture or in tissues of infected animals results in cell death. Cell death of infected neurons and myocytes contributes to the pathogenesis of reovirus-induced encephalitis and myocarditis in a newborn mouse model. Thus, reovirus-induced cell death has been used to investigate the basis of viral disease. Depending on the cell type, infection of host cells by reovirus results in one of two forms of cell death-apoptosis and necroptosis. In addition to the obvious differences in how these two forms of cell death are executed, the mechanisms by which reovirus infection initiates and transduces signals that lead to each of these types of cell death are distinct. In this review, we discuss how apoptosis and necroptosis are triggered by events at different stages of infection. We also describe how innate immune recognition of reovirus genomic material and type I interferon signaling pathways connect with the core components of the apoptosis and necroptosis machinery. The impact of different cell death mediators on viral pathogenesis and the potential of reovirus as an oncolytic vector are also outlined.

Cardiac Contractility Modulation Attenuates Chronic Heart Failure in a Rabbit Model via the PI3K/AKT Pathway.

The Akt plays an important role in regulating cardiac growth, myocardial angiogenesis, and cell death in cardiac myocytes. However, there are few studies to focus on the responses of the Akt pathway to cardiac contractility modulation (CCM) in a chronic heart failure (HF) model. In this study, the effects of CCM on the treatment of HF in a rabbit model were investigated. Thirty six-month-old rabbits were randomly separated into control, HF, and CCM groups. The rabbits in HF and CCM groups were pressure uploaded, which can cause an aortic constriction. Then, CCM was gradually injected to the myocardium of rabbits in the CCM group, and this process lasted for four weeks with six hours per day. Rabbit body weight, heart weight, and heart beating rates were recorded during the experiment. To assess the CCM impacts, rabbit myocardial histology was examined as well. Additionally, western blot analysis was employed to measure the protein levels of Akt, FOXO3, Beclin, Pi3k, mTOR, GSK-3β, and TORC2 in the myocardial histology of rabbits. Results showed that the body and heart weight of rabbits decreased significantly after suffering HF when compared with those in the control group. However, they gradually recovered after CCM application. The CCM significantly decreased collagen volume fraction in myocardial histology of HF rabbits, indicating that CCM therapy attenuated myocardial fibrosis and collagen deposition. The levels of Akt, FOXO3, Beclin, mTOR, GSK-3β, and TORC2 were significantly downregulated, but Pi3k concentration was greatly upregulated after CCM utilization. Based on these findings, it was concluded that CCM could elicit positive effects on HF therapy, which was potentially due to the variation in the Pi3k/Akt signaling pathway.

Impaired NF-κB signalling underlies cyclophilin D-mediated mitochondrial permeability transition pore opening in doxorubicin cardiomyopathy.

The chemotherapy drug doxorubicin (Dox) is commonly used for treating a variety of human cancers; however, it is highly cardiotoxic and induces heart failure. We previously reported that the Bcl-2 mitochondrial death protein Bcl-2/19kDa interaction protein 3 (Bnip3), is critical for provoking mitochondrial perturbations and necrotic cell death in response to Dox; however, the underlying mechanisms had not been elucidated. Herein, we investigated mechanism that drives Bnip3 gene activation and downstream effectors of Bnip3-mediated mitochondrial perturbations and cell death in cardiac myocytes treated with Dox. Nuclear factor-κB (NF-κB) signalling, which transcriptionally silences Bnip3 activation under basal states in cardiac myocytes was dramatically reduced following Dox treatment. This was accompanied by Bnip3 gene activation, mitochondrial injury including calcium influx, permeability transition pore (mPTP) opening, loss of nuclear high mobility group protein 1, reactive oxygen species production, and cell death. Interestingly, impaired NF-κB signalling in cells treated with Dox was accompanied by protein complexes between Bnip3 and cyclophilin D (CypD). Notably, Bnip3-mediated mPTP opening was suppressed by inhibition of CypD-demonstrating that CypD functionally operates downstream of Bnip3. Moreover, restoring IKKβ-NF-κB activity in cardiac myocytes treated with Dox suppressed Bnip3 expression, mitochondrial perturbations, and necrotic cell death. The findings of the present study reveal a novel signalling pathway that functionally couples NF-κB and Dox cardiomyopathy to a mechanism that is mutually dependent upon and obligatorily linked to the transcriptional control of Bnip3. Our findings further demonstrate that mitochondrial injury and necrotic cell death induced by Bnip3 is contingent upon CypD. Hence, maintaining NF-κB signalling may prove beneficial in reducing mitochondrial dysfunction and heart failure in cancer patients undergoing Dox chemotherapy.

Abstract 734: NF-kB Signaling Regulates Mitochondrial Permeability Transition Pore Opening of Cardiac Myocytes via Cyclophilin D (CypD) Modulation

Nuclear Factor-κB (NF-κB) is ubiquitously present transcription factor that regulates a variety of cellular functions including cell survival. Herein, we show a critical role for NF-κB signaling in regulation of mitochondrial permeability transition pore opening (mPTP) of cardiac myocytes that involves cyclophilin D (CypD). Cardiac myocytes expressing a kinase defective form of IKKβ (IKK K-M ), the principle IKK required for NF-κB activation, displayed impaired NF-κB gene activity. Defects in NF-κB signaling coincided with an increase in mPTP opening and cell death. Interestingly, mPTP opening and cell death observed in the NF-κB defective cardiomyocytes was supressed by inhibition of mPTP modulator CypD, with cyclosporin A (CSA) or by siRNA knock down (CypDsiRNA), suggesting a link between mPTP regulation and NF-κB signaling. Earlier, we reported that doxorubicin (Dox) treatment resulted in severe ultra-structural defects including disrupted mitochondrial cristae and impaired respiration, increased mitochondrial calcium overload, mPTP opening and a widespread cell death. Interestingly, we observed a dramatic reduction in NF-κB signaling in cardiac myocytes treated with doxorubicin (18 Hrs), coupled with mitochondrial dysfunction including impaired respiration. Inhibition of CyPD suppressed doxorubicin induced cell death of cardiac myocytes. Finally restoration of NF-κB signaling in cardiac myocytes treated with doxorubicin by IKKβ, active kinase, suppressed mitochondrial calcium overload, mitochondrial perturbations, respiration and cell death. The data herein, provides the first direct evidence that impaired NF-κB signaling predispose Dox treated cardiac myocytes to cell death. Hence, interventions that preserve NF-κB survival signaling pathways in the heart may prove beneficial in reducing cardiac dysfunction and heart failure in cancer patients undergoing doxorubicin chemotherapy.

Effects of Abscisic Acid on the Diabetic Changes in Rat Myocardium

The present study aims to find out the possible protective effect of abscisic acid (ABA) on the development of diabetic cardiomyopathy (DCM) in type 2 diabetic rats. Materials and methods: Fifty male rats divided into: normal control group, diabetic group and three diabetic treated groups with either pioglitazone, or ABA, or both pioglitazone and ABA for 16 weeks. At the end of experiment, ECG was recorded, biochemical measurement of serum glucose, insulin, lipid profile, troponin I, creatine kinase MB (CK-MB), Lactate dehydrogenase (LDH), interleukin-1 beta (IL-1β) and tumor necrosis factor- alpha (TNF-α) was done, caspase 3 and 9 activity in heart, RT- PCR for connexin-43(Cx43) and histopathological examination of cardiac tissue. Results: Treatment with ABA exerted positive effects on blood glucose and insulin levels that found to be reflected on heart weight/body weight ratio in diabetic rats. Also, it exerted significant improvement in cardiac markers and pro-inflammatory cytokines. DCM is associated with increased myocyte cell death that indicated by increasing caspase 3 and 9 that improved significantly by ABA. Also, results indicated that myocardial Cx43 mRNA levels were lesser in diabetic versus non-diabetic rats. Cx43 deterioration in diabetics may be behind the prolongation of the QRS and QTc, that improved by ABA. The histopathological findings showed that ABA improved diabetic cardiomyocyte necrosis and fibrosis. Conclusion: The diabetic rats benefit from ABA intake due to its hypoglycemic, anti-inflammatory and anti-apoptotic effects. So, intake of ABA in combination with anti-diabetic drugs may be beneficial for the management of type 2 diabetes mellitus.

Usefulness of Circulating Caspase-3 p17 and Caspase-1 p20 Peptides and Cardiac Troponin 1 During Cardioplegia to Gauge Myocardial Preservation

Evidence is accumulating that cardiac apoptosis occurs and contributes to myocyte cell death during myocardial ischemia. Cardioplegia, defined as the temporary cessation of cardiac activity during cardiac surgery, is a clinically controlled condition with myocardial ischemia and reperfusion. Our goal was to determine whether the apoptotic biomarker caspase-3 p17 is elevated in the coronary sinus (CS) during cardioplegia and if any elevations were reflected in the peripheral venous (PV) blood. Levels of the necrotic biomarker cardiac troponin I (cTnI) and the inflammatory marker caspase-1 p20 were also quantified in CS and PV. Blood was drawn before and at the end of cardioplegia in PV and CS and levels of p20, p17, and cTnI were measured. cTnI, p20, and p17 PV levels were significantly elevated compared with the control population before and at the end of cardioplegia. PV levels of all 3 markers increased after cardioplegia. CS levels were higher than PV levels for all 3 markers at both time points. Our data are consistent with the occurrence of cardiac apoptosis and inflammation during cardioplegia, in addition to necrosis. The heart-derived markers contributed to the peripheral levels and suggest that measurement of PV biomarker concentrations can be used to gauge cardiac preservation.

Protease-activated receptor 1 activation enhances doxorubicin-induced cardiotoxicity

ObjectiveThe anti-cancer anthracycline drug Doxorubicin (Dox) causes cardiotoxicity. We investigated the role of protease-activated receptor 1 (PAR-1) in Dox-induced cardiotoxicity. Methods and resultsIn vitro experiments revealed that PAR-1 enhanced Dox-induced mitochondrial dysfunction, reactive oxygen species and cell death of cardiac myocytes and cardiac fibroblasts. The contribution of PAR-1 to Dox-induced cardiotoxicity was investigated by subjecting PAR-1−/− mice and PAR-1+/+ mice to acute and chronic exposure to Dox. Heart function was measured by echocardiography. PAR-1−/− mice exhibited significant less cardiac injury and dysfunction compared to PAR-1+/+ mice after acute and chronic Dox administration. PAR-1−/− mice had reduced levels of nitrotyrosine, apoptosis and inflammation in their heart compared to PAR-1+/+ mice. Furthermore, inhibition of PAR-1 in wild-type mice with vorapaxar significantly reduced the acute Dox-induced cardiotoxicity. ConclusionOur results indicate that activation of PAR-1 contributes to Dox-induced cardiotoxicity. Inhibition of PAR-1 may be a new approach to reduce Dox-induced cardiotoxicity in cancer patients.