Search

Mitochondria are important for regulating myocardial physiology, cardiac function and cardio-protection from ischemia/reperfusion injury (IR). Cation channels like BK Ca and K ATP present in the cardiac mitochondria are important in reducing myocardial infarction (MI) upon IR injury. Mitochondrial anion channels are equally important as they regulate cell volume, maintain ionic homeostasis during ischemia and are considered as potential therapeutics for cardiac diseases. However, molecular identity of cardiac mitochondrial chloride channels is not yet elucidated. In this study we focus on unique class of dimorphic ion channels known as chloride intracellular channels (CLICs). Amongst the six paralogs in mammals, we found that CLIC1, CLIC4 and CLIC5 are abundant in heart isolated from R. norvegicus . CLIC4 and CLIC5 localize to the mitochondria whereas CLIC1 in endoplasmic reticulum. CLIC4 and CLIC5 localized to the outer and inner mitochondrial membrane (IMM), respectively, and modulate mitochondrial calcium retention capacity and reactive oxygen species (ROS) production. Interestingly, a splice variant CLIC5B predominantly present in the heart of R. norvegicus was responsible for IMM localization and its function. We also observed that CLICs are cardioprotective as blocking them with its inhibitor IAA-94 increased MI both in in vitro and in vivo IR injury assays . In addition, we performed left main descending coronary artery (LCA) occlusion in clic -/- and wild type (Wt) mice to induce IR injury. LCA was occluded for 40 min prior to 24 h of reperfusion. MI was significantly higher in clic4 -/- mice as compared to Wt mice (58±6 vs 42±2, n≥4, p≤0.05). Only 75% (3 of 4) of clic4 -/- mice showed significant reduction in left ventricular ejection (LVEF) fraction and fractional shortening (LVFS) after in vivo IR injury. Interestingly, 50% (3 of 6) of clic5 -/- mice showed significant increased MI as well as decreased LVEF and LVFS after in vivo IR injury. Clic1 -/- mice showed similar MI and left ventricular function as Wt mice. In conclusion, we demonstrate that cardiac mitochondrial CLIC4 and CLIC5 are vital for cardioprotection from IR injury likely by maintaining the calcium homeostasis and ROS generation by the mitochondria, respectively.

The mitochondrial calcium uniporter channel (mtCU) resides in the inner mitochondrial membrane and is a multi-subunit complex required for mitochondrial Ca 2+ ( m Ca 2+ ) uptake. RNA-seq and PCR analysis of mtCU component expression revealed that MCUB expression increases after myocardial infarction. To investigate how MCUB , a paralog of the MCU gene, contributes to mtCU regulation we created a MCUB -/- cell line (HeLa) using CRISPR-Cas9n. MCUB deletion increased histamine-mediated m [Ca 2+ ] transient amplitude by ~50% vs. WT controls (mito-R-GECO). Biophysical characterization found MCUB deletion increased mtCU capacitance and rate of m [Ca 2+ ] uptake. FPLC fractionation of mtCU revealed loss of MCUB increased MCU incorporation into the supercomplex, suggesting MCUB replacement of MCU and overall increase in functional mtCU supercomplexes. Next, we generated a cardiac-specific, tamoxifen-inducible MCUB overexpression mouse model (CAG-CAT-MCUB x MCM; MCUB-Tg) to examine how the MCUB/MCU ratio regulates mtCU function and cardiac physiology. MCUB-Tg mice were infected with AAV9-mitycam (mito genetic calcium reporter) and adult cardiomyocytes were isolated to record [ m Ca 2+ ] transients during pacing. Increasing the MCUB/MCU ratio decreased [ m Ca 2+ ] peak amplitude by ~30% and significantly reduced the [ m Ca 2+ ] uptake rate. FPLC assessment of the mtCU revealed MCUB was undetectable in the supercomplex fraction (~700kD) of Cre controls, but enriched in MCUB -Tg hearts. Examining the oxygen consumption rate of cardiomyocytes isolated from MCUB- Tg hearts found decreased maximal respiration and spare reserve capacity, which correlated with severe impairment in isoproterenol-induced contractile reserve. Mitochondria from MCUB -Tg mouse hearts were resistant to Ca 2+ induced mitochondrial swelling suggesting MCUB protects against permeability transition. MCUB -Tg mice subjected to myocardial ischemia reperfusion injury had a ~50% decrease in infarct size suggesting increased MCUB expression prevents m Ca 2+ overload and limits cell death. These data suggest that MCUB regulation of the mtCU is an endogenous compensatory mechanism to decrease m Ca 2+ overload during ischemic injury, but maladaptive in mitochondrial energetic responsiveness.

Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself induces further cardiomyocyte death. We recently demonstrated that the pepducin ICL1-9, a small lipidated peptide fragment designed from the first intracellular loop of β2AR, allosterically engaged pro-survival signaling cascades and enhanced cardiomyocyte contractile function in a βarr-dependent manner in vitro. Thus, in this study we tested whether ICL1-9 relays cardioprotection against ischemia/reperfusion (I/R)-induced injury in vivo. Wild-type (WT) C57BL/6 mice received intracardiac injections of either ICL1-9 or a scrambled control pepducin (Scr) at the time of ischemia (30 min) followed by reperfusion for either 24 hours, to assess infarct size and cardiomyocyte death, or 4 weeks, to monitor the impact of ICL1-9 on long-term cardiac structure and function. Intramyocardial injection of ICL1-9 at the time of I/R reduced infarct size, cardiomyocyte death and improved cardiac function in a β2AR- and βarr-dependent manner, which led to less cardiac fibrosis and improved cardiac function over time. Neonatal rat ventricular myocytes (NRVM) were used in conjunction with serum deprivation or hypoxia/reoxygenation (H/R) models to assess the mechanism by which ICL1-9 promotes cardiomyocyte survival. Notably, ICL1-9 attenuated mitochondrial superoxide production and promoted cardiomyocyte survival in a RhoA-dependent manner. ICL1-9 did not alter β2AR density in NRVM or whole heart even up to 24 hr post-treatment, at which timepoint both ICL1-9 localization in cardiomyocytes and RhoA activation were detected, indicating long-lasting presence and effects of ICL1-9 on βarr-dependent β2AR signaling. Thus, βarr-biased β2AR-selective allosteric modulation represents a novel therapeutic approach to reduce reperfusion-induced cardiac injury and relay long-term structural and functional benefits.

Introduction Acute myocardial infarction (MI) is a primary cause of worldwide morbidity and mortality. Macrophages are fundamental components of post-MI inflammation. Pro-inflammatory macrophages can lead to adverse cardiac remodeling and heart failure while anti-inflammatory/reparative macrophages enhance tissue healing. Shifting the balance between pro-inflammatory and reparative macrophages post-MI is a novel therapeutic strategy. Azithromycin (AZM), a commonly used macrolide antibiotic, polarizes macrophages towards the anti-inflammatory phenotype, as shown in animal and human studies. We hypothesized that AZM modulates post-MI inflammation and improves cardiac recovery. Methods and results Male WT mice (C57BL/6, 6–8 weeks old) were treated with either oral AZM (160 mg/kg/day) or vehicle (control) starting 3 days prior to MI and continued to day 7 post-MI. We observed a significant reduction in mortality with AZM therapy. AZM-treated mice showed a significant decrease in pro-inflammatory (CD45+/Ly6G-/F4-80+/CD86+) and increase in anti-inflammatory (CD45+/Ly6G-/F4-80+/CD206+) macrophages, decreasing the pro-inflammatory/anti-inflammatory macrophage ratio in the heart and peripheral blood as assessed by flow cytometry and immunohistochemistry. Macrophage changes were associated with a significant decline in pro- and increase in anti-inflammatory cytokines. Mechanistic studies confirmed the ability of AZM to shift macrophage response towards an anti-inflammatory state under hypoxia/reperfusion stress. Additionally, AZM treatment was associated with a distinct decrease in neutrophil count due to apoptosis, a known signal for shifting macrophages towards the anti-inflammatory phenotype. Finally, AZM treatment improved cardiac recovery, scar size, and angiogenesis. Conclusion Azithromycin plays a cardioprotective role in the early phase post-MI through attenuating inflammation and enhancing cardiac recovery. Post-MI treatment and human translational studies are warranted to examine the therapeutic applications of AZM.

Apoptosis is associated with various myocardial diseases. Angiotensin II (Ang II) plays a central role in the pathogenesis of RAAS‐triggered cardiac apoptosis. Our previous studies showed that mammalian Ste20‐like kinase 1 (Mst1) aggravates cardiac dysfunction in cardiomyocyte under pathological conditions, but its role in Ang II‐mediated cardiomyocyte apoptosis is not known. We addressed this in the present study by investigating whether cardiac‐specific Mst1 knockout can alleviate Ang II‐induced cardiomyocyte apoptosis along with the underlying mechanisms. In vitro and in vivo experiments showed that Ang II increased intracellular reactive oxygen species (ROS) production and cardiomyocyte apoptosis; these were reversed by administration of the ROS scavenger N‐acetylcysteine and by Mst1 deficiency, which suppressed c‐Jun N‐terminal kinase (JNK) phosphorylation and downstream signaling. Interestingly, Mst1 knockout failed to alleviate Ang II‐induced phosphorylation of extracellular signal‐regulated kinase 1/2, and inactivated apoptosis signal‐regulating kinase1 (ASK1) by promoting its association with thioredoxin (Trx), which reversed the Ang II‐induced activation of the ASK1–JNK pathway and suppressed Ang II‐induced cardiomyocyte apoptosis. Thus, cardiac‐specific Mst1 knockout inhibits ROS‐mediated JNK signalling to block Ang II‐induced cardiomyocyte apoptosis, suggesting Mst1 as a potential therapeutic target for treatment of RAAS‐activated heart failure.

Diabetic cardiomyopathy (DCM) is characterized by cardiac microvascular endothelial cells (CMECs) injury and cardiomyocyte (CM) dysfunction. Exosomes mediated cellular communication between CMECs and CM has emerging roles in the pathogenesis of DCM, but the underlining mechanisms are unclear. Mammalian sterile 20-like kinase 1 (Mst1), a key component in Hippo pathway which participates in regulating organ size, apoptosis and autophagy, is involved in the development of DCM. We generated the endothelial-specific Mst1 transgenic mice (Tg-Mst1EC) and constructed diabetic model with streptozotocin (STZ). Interestingly, Tg-Mst1EC mice suffered from worse cardiac function and aggravated insulin resistance compared with non-transgenic (NTg) diabetic mice. The content of Mst1 protein was increased, while Mst1 mRNA had no significant change in CM isolated from diabetic Tg-Mst1EC mice. In vitro, CMECs-derived exosomes were taken up by CM and increased Mst1 protein content which inhibited autophagy, as well as enhanced apoptosis in high glucose (HG) cultured CM as evidenced by immunofluorescence and western blot analysis. In addition, Mst1 inhibited glucose uptake under diabetic condition by disrupting the glucose transporter type 4 (GLUT4) membrane translocation through decreasing the interaction between Daxx and GLUT4, as well as enhancing the association of Mst1 and Daxx. Our study exemplifies pleiotropic effects of Mst1-enriched exosomes released from CMECs on inhibiting autophagy, promoting apoptosis and suppressing the glucose metabolism in CM.

Aims Angiotension II (Ang II) plays a central role in the pathogenesis of renin-angiotensin-aldosterone system (RAAS)-induced heart failure. Mst1 exerts its function in cardiomyocytes subjected to pathological stimuli via inhibiting autophagy and aggravating apoptosis, but its role in RAAS-mediated cardiac injury is still unknown. Here, we aimed to determine whether cardiomyocyte-specific Mst1 knockout can alleviate Ang II-induced cardiac injury by improving cardiomyocyte autophagy and whether these functions depend on Ang II receptors. Results Mst1 knockout alleviated Ang II-induced heart failure, without affecting blood pressure and compensatory concentric hypertrophy. Mst1 specific knockout improved the effects of Ang II on cardiomyocyte autophagy, as evidenced by further increased LC3-II expression and decreased P62 expression. More typical autophagosomes accompanied by less damaged mitochondria were also observed by electron microscopy in Ang II-treated Mst1Δ/Δ mice. In vitro, Mst1 knockdown promoted cardiomyocyte autophagic flux, as demonstrated by more GFP-mRFP-LC3 puncta per cell. Increased LC3-II and decreased P62 expression both in the presence and absence of chloroquine were observed in Mst1 knockdown cardiomyocytes administered with Ang II. Treatment with 3-MA, an inhibitor of autophagy, abolished the beneficial effects of Mst1 knockout against Ang II-induced cardiac dysfunction. The compensatory effects of Ang II on upregulated autophagy were associated with Mst1 inhibition. Interestingly, the knockdown or antagonization of AT1R inhibited cardiomyocyte autophagy, which may represent a threat to cardiac function. Importantly, Mst1 knockout consistently enhanced cardiomyocyte autophagy following the knockdown or blocking of AT1R and AT2R. Conclusion Cardiomyocyte-specific Mst1 knockout alleviates Ang II-induced cardiac injury by enhancing cardiomyocyte autophagy. Mst1 inhibition may counteract the undesirable effects of Ang II receptors blockage on cardiomyocyte autophagy and represent a promising complementary treatment strategy against Ang II-induced cardiac injury.

Engraftment of embryonic stem cells (ESC) has been proposed as a potential therapeutic approach for post-infarction cardiac dysfunction. However, only mild function improvement has been achieved due to low survival rate and paracrine dysfunction of transplanted stem cells. Cellular repressor of E1A stimulated genes (CREG) has been reported to be a secreted glycoprotein implicated in promoting survival and differentiation of many cell types. Therefore we hypothesized that transplantation of genetically modified ESC with CREG (CREG-ESC) can improve cardiac function after myocardial infarction in mice. A total of 2 × 105 CREG-ESC or EGFP-ESC were engrafted into the border zone in a myocardial infarction model in mice. Cardiac function, infarct size and fibrosis at 4 weeks, survival of transplanted ESC, apoptosis and cytokine level of heart tissue, and teratoma formation were assessed in vivo. Apoptosis of ESC under inflammatory stimuli and cardiac differentiation of ESC were investigated in vitro. After 4 weeks, we found transplantation of CREG-ESC could significantly improve cardiac function, ameliorate cardiac remodeling, and reduce infarct size and fibrosis area. Transplantation of CREG-ESC remarkably increased ESC survival in the border zone and inhibited apoptosis of cardiomyocytes. Furthermore, the decrease of inflammatory factors (IL-1β, IL-6 and TNF-α) and increase of anti-inflammatory factors (TGF-β, bFGF and VEGF165) in the border zone were higher in CREG-ESC transplanted hearts. Safety evaluation showed that all transplantation at 2 × 105 per heart dose produced no teratoma. Surprisingly, the mice with 3.0 × 106 CREG-ESC transplantation was demonstrated teratoma free without cardiac rhythm disturbances in contrast to 100% teratoma formation and rhythm abnormality for the same dose of EGFP-ESC transplantation. In addition, overexpression of CREG inhibits ESC apoptosis and enhanced their differentiation into cardiomyocytes in vitro. Transplantation of CREG-modified ESC exhibits a favorable survival pattern in infarcted hearts, which translates into a substantial preservation of cardiac function after acute myocardial infarction.

Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself induces further cardiomyocyte death. β-arrestin (βarr)-biased β-adrenergic receptor (βAR) activation promotes survival signaling responses in vitro; thus, we hypothesize that this pathway can mitigate cardiomyocyte death at the time of reperfusion to better preserve function. However, a lack of efficacious βarr-biased orthosteric small molecules has prevented investigation into whether this pathway relays protection against ischemic injury in vivo. We recently demonstrated that the pepducin ICL1-9, a small lipidated peptide fragment designed from the first intracellular loop of β2AR, allosterically engaged pro-survival signaling cascades in a βarr-dependent manner in vitro. Thus, in this study we tested whether ICL1-9 relays cardioprotection against ischemia/reperfusion (I/R)-induced injury in vivo. Methods: Wild-type (WT) C57BL/6, β2AR knockout (KO), βarr1KO and βarr2KO mice received intracardiac injections of either ICL1-9 or a scrambled control pepducin (Scr) at the time of ischemia (30 min) followed by reperfusion for either 24 h, to assess infarct size and cardiomyocyte death, or 4 weeks, to monitor the impact of ICL1-9 on long-term cardiac structure and function. Neonatal rat ventricular myocytes (NRVM) were used to assess the impact of ICL1-9 versus Scr pepducin on cardiomyocyte survival and mitochondrial superoxide formation in response to either serum deprivation or hypoxia/reoxygenation (H/R) in vitro and to investigate the associated mechanism(s). Results: Intramyocardial injection of ICL1-9 at the time of I/R reduced infarct size, cardiomyocyte death and improved cardiac function in a β2AR- and βarr-dependent manner, which led to improved contractile function early and less fibrotic remodeling over time. Mechanistically, ICL1-9 attenuated mitochondrial superoxide production and promoted cardiomyocyte survival in a RhoA/ROCK-dependent manner. RhoA activation could be detected in cardiomyocytes and whole heart up to 24 h post-treatment, demonstrating the stability of ICL1-9 effects on βarr-dependent β2AR signaling. Conclusion: Pepducin-based allosteric modulation of βarr-dependent β2AR signaling represents a novel therapeutic approach to reduce reperfusion-induced cardiac injury and relay long-term cardiac remodeling benefits.

Caspase-1 activation senses metabolic danger-associated molecular patterns (DAMPs) and mediates the initiation of inflammation in endothelial cells. Here, we examined whether the caspase-1 pathway is responsible for sensing hyperlipidemia as a DAMP in bone marrow (BM)-derived Stem cell antigen-1 positive (Sca-(1+)) stem/progenitor cells and weakening their angiogenic ability. Using biochemical methods, gene knockout, cell therapy and myocardial infarction (MI) models, we had the following findings: 1) Hyperlipidemia induces caspase-1 activity in mouse Sca-(1+) progenitor cells in vivo; 2) Caspase-1 contributes to hyperlipidemia-induced modulation of vascular cell death-related gene expression in vivo; 3) Injection of Sca-1+ progenitor cells from caspase-1(-/-) mice improves endothelial capillary density in heart and decreases cardiomyocyte death in a mouse model of MI; and 4) Caspase-1(-/-) Sca-(1+) progenitor cell therapy improves mouse cardiac function after MI. Our results provide insight on how hyperlipidemia activates caspase-1 in Sca-(1+) progenitor cells, which subsequently weakens Sca-(1+) progenitor cell repair of vasculature injury. These results demonstrate the therapeutic potential of caspase-1 inhibition in improving progenitor cell therapy for MI.

Background The innate immune receptor NLRP3 recognizes tissue damage and initiates inflammatory processes through formation multiprotein complexes with the adaptor protein ASC and caspase-1, i.e. NLRP3 inflammasomes, which through cleavage of pro-IL-1β mediates release of bioactive IL-1β. We hypothesized that NLRP3 mediates tissue damage during acute myocardial infarction (MI) and sought to investigate the mechanisms herein in an experimental MI model in mice. Methods and results The left coronary artery (LCA) of WT, NLRP3 -/- and ASC -/- mice of both genders was ligated for 30 min followed by 3 or 24 h reperfusion. For pre-conditioning studies, the TLR2 agonist Pam3CSK4 or PBS was injected intraperitoneally 60 min prior to LCA ligation. For mechanistic investigations, blood plasmas and left ventricle tissues were collected, and a hypothesis-driven selection of protein or mRNA targets was investigated. Surprisingly, hearts from NLRP3-deficient mice featured larger infarct size than WT mice (p = 0.0048). In general, there were only modest changes with no significant pattern in myocardial infiltration of neutrophils and macrophages and systemic and myocardial cytokine expression between the three genotypes. Preconditioning with the TLR2 agonist Pam3CSK4 induced Akt phosphorylation and reduced infarct size in WT but not NLRP3 -or ASC -deficient hearts. Conclusion Absence of NLRP3 results in increased myocardial infarct size after in vivo ischemia reperfusion, seemingly due to dysfunction of the cardioprotective RISK pathway. Our data imply that NLRP3 contributes to cardio-protection during I/R and do not support a role for NLRP3 or ASC inhibition in the management of acute MI including revascularization therapy.

Rationale : G protein–coupled receptor kinases (GRKs) are dynamic regulators of cellular signaling. GRK5 is highly expressed within myocardium and is upregulated in heart failure. Although GRK5 is a critical regulator of cardiac G protein–coupled receptor signaling, recent data has uncovered noncanonical activity of GRK5 within nuclei that plays a key role in pathological hypertrophy. Targeted cardiac elevation of GRK5 in mice leads to exaggerated hypertrophy and early heart failure after transverse aortic constriction (TAC) because of GRK5 nuclear accumulation. Objective : In this study, we investigated the role of GRK5 in physiological, swimming-induced hypertrophy (SIH). Methods and Results : Cardiac-specific GRK5 transgenic mice and nontransgenic littermate control mice were subjected to a 21-day high-intensity swim protocol (or no swim sham controls). SIH and specific molecular and genetic indices of physiological hypertrophy were assessed, including nuclear localization of GRK5, and compared with TAC. Unlike after TAC, swim-trained transgenic GRK5 and nontransgenic littermate control mice exhibited similar increases in cardiac growth. Mechanistically, SIH did not lead to GRK5 nuclear accumulation, which was confirmed in vitro as insulin-like growth factor-1, a known mediator of physiological hypertrophy, was unable to induce GRK5 nuclear translocation in myocytes. We found specific patterns of altered gene expression between TAC and SIH with GRK5 overexpression. Further, SIH in post-TAC transgenic GRK5 mice was able to preserve cardiac function. Conclusions: These data suggest that although nuclear-localized GRK5 is a pathological mediator after stress, this noncanonical nuclear activity of GRK5 is not induced during physiological hypertrophy.

The orphan nuclear receptor Nur77 plays critical roles in cardiovascular diseases, and its expression is markedly induced in the heart after beta-adrenergic receptor (β-AR) activation. However, the functional significance of Nur77 in β-AR signaling in the heart remains unclear. By using Northern blot, Western blot, and immunofluorescent staining assays, we showed that Nur77 expression was markedly upregulated in cardiomyocytes in response to multiple hypertrophic stimuli, including isoproterenol (ISO), phenylephrine (PE), and endothelin-1 (ET-1). In a time- and dose-dependent manner, ISO increases Nur77 expression in the nuclei of cardiomyocytes. Overexpression of Nur77 markedly inhibited ISO-induced cardiac hypertrophy by inducing nuclear translocation of Nur77 in cardiomyocytes. Furthermore, cardiac overexpression of Nur77 by intramyocardial injection of Ad-Nur77 substantially inhibited cardiac hypertrophy and ameliorated cardiac dysfunction after chronic infusion of ISO in mice. Mechanistically, we demonstrated that Nur77 functionally interacts with NFATc3 and GATA4 and inhibits their transcriptional activities, which are critical for the development of cardiac hypertrophy. These results demonstrate for the first time that Nur77 is a novel negative regulator for the β-AR-induced cardiac hypertrophy through inhibiting the NFATc3 and GATA4 transcriptional pathways. Targeting Nur77 may represent a potentially novel therapeutic strategy for preventing cardiac hypertrophy and heart failure.

Adiponectin (APN) is a cardioprotective molecule. Its reduction in diabetes exacerbates myocardial ischemia/reperfusion (MI/R) injury. Although APN administration in animals attenuates MI/R injury, multiple factors limit its clinical application. The current study investigated whether AdipoRon, the first orally active molecule that binds APN receptors, may protect the heart against MI/R injury, and if so, to delineate the involved mechanisms. Wild-type (WT), APN knockout (APN-KO), and cardiomyocyte specific-AMPK dominant negative (AMPK-DN) mice were treated with vehicle or AdipoRon (50 mg/kg, 10 min prior to MI) and subjected to MI/R (30 min/3–24 h). Compared with vehicle, oral administration of AdipoRon to WT mice significantly improved cardiac function and attenuated postischemic cardiomyocyte apoptosis, determined by DNA ladder formation, TUNEL staining, and caspase-3 activation (all P < 0.01). MI/R-induced apoptotic cell death was significantly enhanced in mice deficient in either APN (APN-KO) or AMPK (AMPK-DN). In APN-KO mice, AdipoRon attenuated MI/R injury to the same degree as observed in WT mice. In AMPK-DN mice, AdipoRon's antiapoptotic action was partially inhibited but not lost. Finally, AdipoRon significantly attenuated postischemic oxidative stress, as evidenced by reduced NADPH oxidase expression and superoxide production. Collectively, these results demonstrate for the first time that AdipoRon, an orally active APN receptor activator, effectively attenuated postischemic cardiac injury, supporting APN receptor agonists as a promising novel therapeutic approach treating cardiovascular complications caused by obesity-related disorders such as type 2 diabetes.

Since highly active antiretroviral therapy improved long-term survival of acquired immunodeficiency syndrome (AIDS) patients, AIDS cardiomyopathy has become an increasingly relevant clinical problem. We used human immunodeficiency virus (HIV)-1 transgenic (Tg26) mouse to explore molecular mechanisms of AIDS cardiomyopathy. Tg26 mice had significantly lower left ventricular (LV) mass and smaller end-diastolic and end-systolic LV volumes. Under basal conditions, cardiac contractility and relaxation and single myocyte contraction dynamics were not different between wild-type (WT) and Tg26 mice. Ten days after open heart surgery, contractility and relaxation remained significantly depressed in Tg26 hearts, suggesting that Tg26 mice did not tolerate surgical stress well. To simulate heart failure in which expression of Bcl2-associated athanogene 3 (BAG3) is reduced, we down-regulated BAG3 by small hairpin ribonucleic acid in WT and Tg26 hearts. BAG3 down-regulation significantly reduced contractility in Tg26 hearts. BAG3 overexpression rescued contractile abnormalities in myocytes expressing the HIV-1 protein Tat. We conclude: (i) Tg26 mice exhibit normal contractile function at baseline; (ii) Tg26 mice do not tolerate surgical stress well; (iii) BAG3 down-regulation exacerbated cardiac dysfunction in Tg26 mice; (iv) BAG3 overexpression rescued contractile abnormalities in myocytes expressing HIV-1 protein Tat; and (v) BAG3 may occupy a role in pathogenesis of AIDS cardiomyopathy.

Restoring expression levels of the EF-hand calcium (Ca(2+)) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca(2+) handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca(2+) resequestration appears critical for S100A1's cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca(2+) leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF. Restoring this link in failing cardiomyocytes, engineered heart tissue and mouse hearts, respectively, by means of adenoviral and adeno-associated viral S100A1 cDNA delivery normalizes diastolic RyR2 function and protects against Ca(2+)- and β-adrenergic receptor-triggered proarrhythmogenic SR Ca(2+) leakage in vitro and in vivo. S100A1 inhibits diastolic SR Ca(2+) leakage despite aberrant RyR2 phosphorylation via protein kinase A and calmodulin-dependent kinase II and stoichiometry with accessory modulators such as calmodulin, FKBP12.6 or sorcin. Our findings demonstrate that S100A1 is a regulator of diastolic RyR2 activity and beneficially modulates diastolic RyR2 dysfunction. S100A1 interaction with the RyR2 is sufficient to protect against basal and catecholamine-triggered arrhythmic SR Ca(2+) leak in HF, combining antiarrhythmic potency with chronic inotropic actions.

Tumor necrosis factor-α (TNF-α) antagonism alleviates myocardial ischemia-reperfusion (MI/R) injury. However, the mechanisms by which the downstream mediators of TNF-α change after acute antagonism during MI/R remain unclear. Adiponectin (APN) exerts anti-ischemic effects, but it is downregulated during MI/R. This study was conducted to investigate whether TNF-α is responsible for the decrease of APN, and whether antagonizing TNF-α affects MI/R injury by increasing APN. Male adult wild-type (WT), APN knockout (APN KO) mice, and those with cardiac knockdowns of APN receptors via siRNA injection were subjected to 30 min of MI followed by reperfusion. The TNF-α antagonist etanercept or globular domain of APN (gAD) was injected 10 min before reperfusion. Etanercept ameliorated MI/R injury in WT mice as evidenced by improved cardiac function, and reduced infarct size and cardiomyocyte apoptosis. APN concentrations were augmented in response to etanercept, followed by an increase in AMP-activated protein kinase phosphorylation. Etanercept still increased cardiac function and reduced infarct size and apoptosis in both APN KO and APN receptors knockdown mice. However, its potential was significantly weakened in these mice compared with the WT mice. TNF-α is responsible for the decrease in APN during MI/R. The cardioprotective effects of TNF-α neutralization are partially due to the upregulation of APN. The results provide more insight into the TNF-α-mediated signaling effects during MI/R and support the need for clinical trials to validate the efficacy of acute TNF-α antagonism in the treatment of MI/R injury.

TNF-α inhibitor reportedly protects against myocardial ischemia/reperfusion (MI/R) injury. It can also increase Notch1 expression in inflammatory bowel disease, revealing the regulation of Notch1 signaling by TNF-α inhibitor. However, the interaction between TNF-α inhibitor and Notch1 signaling in MI/R remains unclear. This study aimed to determine the involvement of TNF-α inhibitor with Notch1 in MI/R and delineate the related mechanism. Notch1-specific small interfering RNA (20 μg) or Jagged1 (a Notch ligand, 12 μg) was delivered through intramyocardial injection. Forty-eight hours after injection, mice received 30 min of myocardial ischemia followed by 3 h (for cell apoptosis and oxidative/nitrative stress) or 24h (for infarct size and cardiac function) of reperfusion. Ten minutes before reperfusion, mice randomly received an intraperitoneal injection of vehicle, etanercept, diphenyleneiodonium, 1400W, or EUK134. Finally, downregulation of Notch1 significantly reversed the alleviation of MI/R injury induced by etanercept, as evidenced by enlarged myocardial infarct size, suppressed cardiac function, and increased myocardial apoptosis. Moreover, Notch1 blockade increased the expression of inducible NO synthase (iNOS) and gp(91)(phox), enhanced NO and superoxide production, and accelerated their cytotoxic reaction product, peroxynitrite. Furthermore, NADPH inhibition with diphenyleneiodonium or iNOS suppression with 1400W mitigated the aggravation of MI/R injury induced by Notch1 downregulation in mice treated with etanercept. Additionally, either Notch1 activation with Jagged1 or peroxynitrite decomposition with EUK134 reduced nitrotyrosine content and attenuated MI/R injury. These data indicate that MI/R injury can be attenuated by TNF-α inhibitor, partly via Notch1 signaling-mediated suppression of oxidative/nitrative stress.

Background— Phosphorylative desensitization of G-protein–coupled receptors contributes significantly to post–myocardial infarction (MI) remodeling and heart failure (HF). Here, we determined whether adiponectin receptors (AdipoRs) 1 and 2 (the 7-transmembrane domain–containing receptors mediating adiponectin functions) are phosphorylatively modified and functionally impaired after MI. Methods and Results— Post-MI HF was induced by coronary artery occlusion. Receptor phosphorylation, kinase expression, and adiponectin function were determined via in vivo, ex vivo, and in vitro models. AdipoR1 and AdipoR2 are not phosphorylated in the normal heart. However, AdipoR1 was significantly phosphorylated after MI, peaking at 7 days and remaining significantly phosphorylated thereafter. The extent of post-MI AdipoR1 phosphorylation positively correlated with the expression level of GPCR kinase (GRK) 2, the predominant GRK isoform upregulated in the failing heart. Cardiac-specific GRK2 knockout virtually abolished post-MI AdipoR1 phosphorylation, whereas virus-mediated GRK2 overexpression significantly phosphorylated AdipoR1 and blocked adiponectin metabolic-regulatory/anti-inflammatory signaling. Mass spectrometry identified serine-7, threonine-24, and threonine-53 (residues located in the n-terminal intracellular AdipoR1 region) as the GRK2 phosphorylation sites. Ex vivo experiments demonstrated that adenosine monophosphate–activated protein kinase activation and the anti–tumor necrosis factor-α effect of adiponectin were significantly inhibited in cardiomyocytes isolated from nonischemic area 7 days after MI. In vivo experiments demonstrated that acute adiponectin administration–induced cardiac GLUT4 translocation and endothelial nitric oxide synthase phosphorylation were blunted 7 days after MI. Continuous adiponectin administration beginning 7 days after MI failed to protect the heart from adverse remodeling and HF progression. Finally, cardiac-specific GRK2 knockdown restored the cardioprotective effect of adiponectin. Conclusion— AdipoR1 is phosphorylatively modified and desensitized by GRK2 in failing cardiomyocytes, contributing to post-MI remodeling and HF progression.

Ubiquitously expressed Trpm2 channel limits oxidative stress and preserves mitochondrial function. We first demonstrated that intracellular Ca2+concentration increase after Trpm2 activation was due to direct Ca2+influx and not indirectly via reverse Na+/Ca2+exchange. To elucidate whether Ca2+entry via Trpm2 is required to maintain cellular bioenergetics, we injected adenovirus expressing green fluorescent protein (GFP), wild-type (WT) Trpm2, and loss-of-function (E960D) Trpm2 mutant into left ventricles of global Trpm2 knockout (gKO) or WT hearts. Five days post-injection, gKO-GFP heart slices had higher reactive oxygen species (ROS) levels but lower oxygen consumption rate (OCR) than WT-GFP heart slices. Trpm2 but not E960D decreased ROS and restored OCR in gKO hearts back to normal levels. In gKO myocytes expressing Trpm2 or its mutants, Trpm2 but not E960D reduced the elevated mitochondrial superoxide (O2.−) levels in gKO myocytes. After hypoxia-reoxygenation (H/R), Trpm2 but not E906D or P1018L (inactivates Trpm2 current) lowered O2.−levels in gKO myocytes and only in the presence of extracellular Ca2+, indicating sustained Ca2+entry is necessary for Trpm2-mediated preservation of mitochondrial function. After ischemic-reperfusion (I/R), cardiac-specific Trpm2 KO hearts exhibited lower maximal first time derivative of LV pressure rise (+dP/d t) than WT hearts in vivo. After doxorubicin treatment, Trpm2 KO mice had worse survival and lower +dP/d t. We conclude 1) cardiac Trpm2-mediated Ca2+influx is necessary to maintain mitochondrial function and protect against H/R injury; 2) Ca2+influx via cardiac Trpm2 confers protection against H/R and I/R injury by reducing mitochondrial oxidants; and 3) Trpm2 confers protection in doxorubicin cardiomyopathy.

Aims: To determine the roles of vitamin D receptor (VDR) in ischemia/reperfusion-induced myocardial injury and to investigate the underlying mechanisms involved. Results: The endogenous VDR expression was detected in the mouse heart, and myocardial ischemia/reperfusion (MI/R) upregulated VDR expression. Activation of VDR by natural and synthetic agonists reduced myocardial infarct size and improved cardiac function. Mechanistically, VDR activation inhibited endoplasmic reticulum (ER) stress (determined by the reduction of CCAAT/enhancer-binding protein homologous protein expression and caspase-12 activation), attenuated mitochondrial impairment (determined by the decrease of mitochondrial cytochrome c release and caspase-9 activation), and reduced cardiomyocyte apoptosis. Furthermore, VDR activation significantly inhibited MI/R-induced autophagy dysfunction (determined by the inhibition of Beclin 1 over-activation, the reduction of autophagosomes, the LC3-II/LC3-I ratio, p62 protein abundance, and the restoration of autophagy flux). Moreover, VDR activation inhibited MI/R-induced oxidative stress through a metallothionein-dependent mechanism. The cardioprotective effects of VDR agonists mentioned earlier were impaired in the setting of cardiac-specific VDR silencing. In contrast, adenovirus-mediated cardiac VDR overexpression decreased myocardial infarct size and improved cardiac function through attenuating oxidative stress, and inhibiting apoptosis and autophagy dysfunction. Innovation and Conclusion: Our data demonstrate that VDR is a novel endogenous self-defensive and cardioprotective receptor against MI/R injury, via mechanisms (at least in part) reducing oxidative stress, and inhibiting apoptosis and autophagy dysfunction-mediated cell death. Antioxid. Redox Signal. 22, 633–650.

Background Bone mesenchymal stem cell (BMSC) therapy has modest success in ischemic heart disease but has been limited by poor survival in diseased microenvironments. Cellular repressor of E1A-stimulated genes (CREG) can prevent BMSCs from apoptosis in vitro ; however, the effects of CREG-modified BMSCs on ischemic heart disease and the related mechanism remain undefined. Therefore, we designed to study the cardioprotective effects of CREG overexpression in BMSCs ( CREG BMSCs) after transplantation into infarcted heart of rats. Methods In vivo studies, 50μl PBS or 1.5×10 6 Norm BMSCs, GFP BMSCs or CREG BMSCs were implanted intramyocardially in myocardial infarction rat models. 3 or 14days later, cardiac function, fibrosis, apoptosis and angiogenesis were analyzed by echocardiography, masson, western blot and immunofluorescence staining, respectively. ELISA, western blot and matrigel assay were used in vitro to detect vascular endothelial growth factor (VEGF) secretion, signaling molecule expression, and angiogenic tube formation. Results In vivo , prolonged cardiac function (14d LVEF: 50.87±0.94%; LVFS: 23.41±1.12%), decreased fibrosis (14d Fibrotic area: 27.37±1.03%) and apoptosis and increased angiogenesis were observed in CREG BMSCs, compared with other groups. In vivo and in vitro , VEGF secretion from CREG BMSCs was markedly enhanced. In vitro , angiogenic tube formation in CREG BMSC supernatants significantly increased. Moreover, CREG activated hypoxia-inducible factor-1α (HIF-1α), but not HIF-1β. Knockdown of HIF-1α with siRNA decreased VEGF secretion and angiogenic tube formation. Notably, CREG did not influence HIF-1α mRNA synthesis but inhibited the expression of Von Hippel–Lindau (VHL), a key protein that regulates HIF-1α degradation. Conclusions The CREG BMSC transplantation, directly or indirectly, may promote VEGF's anti-apoptosis and angiogenesis via the inhibition of VHL-mediated HIF-1α degradation, consequently protecting against myocardial infarction.

Coronary heart disease patients with type 2 diabetes were subject to higher vulnerability for cardiac ischemia-reperfusion (I/R) injury. This study was designed to evaluate the impact of ZP2495 (a glucagon-GLP-1 dual-agonist) on cardiac function and energy metabolism after myocardial I/R injury in db/db mice with a focus on mitochondrial function. C57BLKS/J-lepr+/lepr+ (BKS) and db/db mice received 4-week treatment of glucagon, ZP131 (GLP-1 receptor agonist), or ZP2495, followed by cardiac I/R injury. The results showed that cardiac function, cardiac glucose metabolism, cardiomyocyte apoptosis, cardiac mitochondrial morphology, and energetic transition were improved or ameliorated by ZP2495 to a greater extent than that of glucagon and ZP131. In vitro study showed that ZP2495, rather than glucagon, alleviated mitochondrial depolarization, cytochrome C release, and mitochondria ROS generation in neonatal rat ventricular myocytes subjected to high-glucose and simulated I/R injury conditions, the effects of which were weaker in the ZP131 group. Furthermore, the expressions of Akt, FoxO3a, and AMPK phosphorylation were elevated by ZP2495 to a greater extent than that of ZP131. In conclusion, ZP2495 may contribute to the improvement of cardiac function and energy metabolism in db/db mice after myocardial I/R injury by improving mitochondrial function possibly through Akt/FoxO3a and AMPK/FoxO3a signal pathways.

The insulin-PI3K-mTOR pathway exhibits a variety of cardiovascular activities including protection against I/R injury. Lin28a enhanced glucose uptake and insulin-sensitivity via insulin-PI3K-mTOR signalling pathway. However, the role of lin28a on experimental cardiac I/R injury in diabetic mice are not well understood. Diabetic mice underwent 30 min. of ischaemia followed by 3 hrs of reperfusion. Animals were randomized to be treated with lentivirus carrying lin28a siRNA (siLin28a) or lin28a cDNA (Lin28a) 72 hrs before coronary artery ligation. Myocardial infarct size (IS), cardiac function, cardiomyocyte apoptosis and mitochondria morphology in diabetic mice who underwent cardiac I/R injury were compared between groups. The target proteins of lin28a were examined by western blot analysis. Lin28a overexpression significantly reduced myocardial IS, improved LV ejection fraction (LVEF), decreased myocardial apoptotic index and alleviated mitochondria cristae destruction in diabetic mice underwent cardiac I/R injury. Lin28a knockdown exacerbated cardiac I/R injury as demonstrated by increased IS, decreased LVEF, increased apoptotic index and aggravated mitochondria cristae destruction. Interestingly, pre-treatment with rapamycin abolished the beneficial effects of lin28a overexpression. Lin28a overexpression increased, while Lin28a knockdown decreased the expression of IGF1R, p-Akt, p-mTOR and p-p70s6k after cardiac I/R injury in diabetic mice. Rapamycin pre-treatment abolished the effects of increased p-mTOR and p-p70s6k expression exerted by lin28a overexpression. This study indicates that lin28a overexpression reduces IS, improves cardiac function, decreases cardiomyocyte apoptosis index and alleviates cardiomyocyte mitochondria impairment after cardiac I/R injury in diabetic mice. The mechanism responsible for the effects of lin28a is associated with the insulin-PI3K-mTOR dependent pathway.

Heart failure (HF) is a leading cause of death worldwide and a growing burden on public health, and the underlying mechanisms of cardiac remodeling and decompensation to HF remain a focus of research efforts towards therapeutic development. Signaling via G protein-coupled receptors (GPCRs) is critical for normal heart function and is tightly controlled by GPCR kinases (GRKs) with GRK2 (originally βARK1), being intimately involved in HF progression. In addition to its well-characterized role in regulating GPCRs, ongoing research has demonstrated great diversity in the functional roles of GRK2. I have recently investigated GRK2 amino terminal binding interactions through the generation of transgenic (Tg) mice with cardiac-targeted expression of the amino-terminal peptide βARKnt (residues 50-145). In a murine model of trans-aortic constriction (TAC)-induced pressure overload, echocardiography revealed increased left ventricular (LV) posterior wall thickness (1.57 versus 1.37 ± 0.02; n = 16) and LV mass in TgβARKnt compared to non-transgenic littermate controls (NLC) 4 weeks after TAC or Sham surgery. Interestingly, despite enhanced hypertrophy at baseline and after acute pressure overload, the progression to HF was paradoxically inhibited in TgβARKnt mice during chronic pressure overload with preserved cardiac function (% Ejection Fraction 57.3 versus 37.3 ± 2.0; n = 11, 10). Further, βARKnt expression limited adverse left ventricular remodeling, with reduced interstitial fibrosis (% area fibrosis 4.1 versus 9.2 ± 0.8; n = 11, 9 hearts) and preserved β-adrenergic receptor density 4 weeks after surgery. The effect of cardiac βARKnt expression was not consistent with alterations in GRK2 activity at GPCRs as neither GRK2 peptide inhibition (TgβARKct) nor GRK2 overexpression alter hypertrophy, and overexpression hastens HF development. Further, TgβARKnt mice exhibit reduced epididymal white adipose content and altered mitochondrial respiration, suggesting altered cardiac metabolism. These data support the idea that the βARKnt peptide embodies a distinct functional interaction and that βARKnt-mediated regulation of β-adrenergic receptor density may provide a novel means of cardioprotection during pressure-overload induced HF.

Aims The farnesoid X receptor (FXR) is a member of the metabolic nuclear receptor superfamily that plays a critical regulatory role in cardiovascular physiology/pathology. However, the role of systemic FXR activation in the chronic phase in myocardial infarction (MI)-induced cardiac remodelling and dysfunction remains unclear. In this study, we aimed to elucidate the role of long-term FXR activation on post-MI cardiac remodelling and dysfunction. Methods and results Mice underwent either MI surgery or sham operation. At 1 week after MI, both sham and MI mice were gavaged with 25 mg/kg/d of a synthetic FXR agonist (GW4064) or a vehicle control for 7 weeks, and cardiac performance was assessed by consecutive echocardiography studies. Administration of GW4064 significantly increased left ventricular ejection fraction at 4 weeks and 8 weeks after MI (both P

Autophagy is increasingly recognized as an important regulator of myocardial ischemia-reperfusion (MI-R) injury. However, whether and how diabetes may alter autophagy in response to MI-R remains unknown. Deficiency of adiponectin (AdipoQ), a cardioprotective molecule, markedly increases MI-R injury. However, the role of diabetic hypoadiponectinemia in cardiac autophagy alteration after MI-R is unclear. Utilizing normal control (NC), high-fat-diet-induced diabetes, and Adipoq knockout ( adipoq -/- ) mice, we demonstrated that autophagosome formation was modestly inhibited and autophagosome clearance was markedly impaired in the diabetic heart subjected to MI-R. adipoq -/- largely reproduced the phenotypic alterations observed in the ischemic-reperfused diabetic heart. Treatment of diabetic and adipoq -/- mice with AdipoRon, a novel adiponectin receptor agonist, stimulated autophagosome formation, markedly increased autophagosome clearance, reduced infarct size, and improved cardiac function (P

Recent studies highlighted that circular RNAs (circRNA) can play an important role in cardiac hypertrophy. However, the circRNAs in cardiac diseases is still limited. Using global circRNA expression profiling, we identified several circRNA transcripts that were differentially regulated post-MI in mice, including mmu_circ_008396 that is significantly down regulated. Cell fractionation experiments indicated that mmu_circ_008396 is highly enriched in endothelial cells in post-MI mice. Interestingly, we found a mmu_circ_008396 circRNA ortholog in humans, which was also significantly down regulated in ischemic cardiomyopathy patients. Further, overexpression of mmu_circ_008396 significantly enhanced tube formation and reduced apoptosis of human umbilical vein endothelial cells. For cardiac overexpression of mmu_circ_008396 circRNA, we created AAV9 viral particles and found that in vivo over expression attenuated LV dysfunction post-MI and enhanced neovascularization. Mechanistically, mmu_circ_008396 binds to its potential target miRNAs (mmu-miR-93-3p, mmu-miR-412-3p and mmu-miR-298-5p) and regulate hemeoxygenase-1/ VEGF signaling, thereby enhancing neovascularization and cardiac repair post-MI. These results indicate that mmu_circ_008396 circRNA might be a novel potential target to prevent cardiac remodeling and also highlight the significance of circRNAs in cardiovascular diseases.

Cell therapy remains the most promising approach against ischemic heart failure. However, the poor survival of engrafted stem cells in the ischemic environment limits their therapeutic efficacy for cardiac repair post-MI. CTRP9 is a novel pro-survival cardiokine with significantly downregulated expression after MI. Here, we tested a hypothesis that CTRP9 might be a cardiokine required for a healthy microenvironment promoting stem cell survival and cardioprotection. Mice were subjected to MI and treated with adipose-derived mesenchymal stem cells (ADSCs, intramyocardial transplantation), CTRP9, or their combination. Administration of ADSCs alone failed to exert significant cardioprotection. However, administration of ADSCs in addition to CTRP9 further enhanced the cardioprotective effect of CTRP9 (P

The mitochondrial calcium uniporter (MCU) is a high-capacity, inward-rectifying channel in the inner mitochondrial membrane and is required for mitochondrial Ca 2+ ( m Ca 2+ ) uptake. m Ca 2+ signaling regulates bioenergetics and activates the mitochondrial permeability transition pore (MPTP) which are cellular processes implicated in cardiac pathophysiology warranting further research into the molecular regulation of the MCU. Recently, a MCU gene paralog, MCUB , was identified as a possible component of the channel. To investigate MCUB’s contribution to uniporter regulation we created a MCUB -/- HeLa cell line using CRISPR-Cas9n. Here, we report that loss of MCUB increased m Ca 2+ transient amplitude after IP3R stimulation (52% vs. con) suggesting MCUB negatively regulates m Ca 2+ uptake. Mitoplast patch-clamping confirmed that loss of MCUB increases MCU current density, suggesting MCUb modulates channel capacitance. Permeabilized MCUB -/- and WT cells exposed to various levels of Ca 2+ (0.5-20μM) revealed that MCUB -/- cells exhibited m Ca 2+ uptake at lower Ca 2+ concentrations than controls, suggesting MCUB contributes to channel gating. In m Ca 2+ retention capacity experiments MCUB -/- cells required ~30% less bath Ca 2+ to induce depolarization, suggesting a predisposition to m Ca 2+ overload. Next, we generated a cardiac-specific, tamoxifen-inducible MCUB overexpression mouse model ( MCUB -Tg). Cardiomyocytes isolated from MCUB -Tg hearts exhibited decreased m Ca 2+ uptake at low-Ca 2+ (59% vs. con) and isolated mitochondria exhibited a reduction in Ca 2+ -induced swelling (37% vs. con), suggesting a resistance to permeability transition. MCUB -Tg mice displayed a significant impairment in isoproterenol-induced contractile reserve and this correlated with a loss of isoproterenol-mediated activation of pyruvate dehydrogenase. In summary, our results suggest that MCUB limits m Ca 2+ uptake by altering channel-gating and thereby regulates bioenergetics and MPTP opening.

Rationale: Coronary artery disease and subsequent myocardial ischemia (MI) are the most common cause of heart failure (HF) in the US. G protein-coupled receptor (GPCR) kinase 5 (GRK5) has been shown to be upregulated in failing human myocardium. While the canonical role of GRKs is to desensitize receptors via phosphorylation, it has been shown that GRK5 can also locate to the nucleus of cardiomyocytes where it can exert GPCR-independent effects that promote maladaptive cardiac hypertrophy after hypertrophic stress. Despite numerous data indicating the importance of GRK5 in hypertrophy, it is still unknown if GRK5 has a role in ischemic heart disease. Objective: In this study, we investigated the critical role that GRK5 plays after myocardial ischemic injury with a novel aspect being the regulation of immune and inflammatory responses including recruitment of immune cells to the injured heart. Methods and Results: Cardiac-specific GRK5 transgenic mice (Tg-GRK5) and non-transgenic littermate (NLC) control mice were subjected to MI. Tg-GRK5-HF mice showed decreased cardiac function (both global and segmental contractility) as well as augmented left ventricular diameters and volumes compared to NLC-HF mice. Heart weight (HW) to body weight or tibia length ratios as well as mRNA expression of all major adverse remodeling-associated biomarkers (ANF, BNP, b-MHC) were increased in the TgGRK5-HF compared to NLC-HF. Cardiac fibrosis in the border zone (BZ) area as well as mRNA levels of Collagen-1 (Col-1), Col-3, MMP2 and CTGF were higher in TgGRK5-HF compared to NLC-HF, strongly suggesting increased adverse remodeling. Inflammatory Cytokines (IL-6 and IL-1beta) were augmented in TgGRK5-HF compared to NLC-HF and contribute at least in part to increased immune cell recruitment in the heart. In fact, we found both Neutrophils and T-lymphocytes (T- cells) augmented in the BZ and infarct zone. Conclusions: Our study shows that cardiac GRK5 has a detrimental effect during ischemic HF. GRK5 overexpression causes reduced cardiac function and increased immune cell recruitment/inflammation. Further, these results suggest GRK5 as a potential therapeutic target to limit HF development after ischemic injury.

β-Adrenergic receptors (βAR) are important regulators of cardiac function in the normal and failing heart. Activation of the β1AR subtype increases contractility and cardiomyocyte death whereas the β2AR can promote survival. In response to cardiac injury, increased catecholamines activate and downregulate β1AR thus, β-adrenergic receptor antagonists or β-blockers are commonly prescribed in to restore β1AR expression and preserve contractility. However, promoting the pro-survival effects of β2AR, which are known to occur in part through β-arrestin (ARR)-dependent signaling, may be a beneficial therapeutic strategy. Pepducins have been developed based on the intracellular loop (ICL) domains of β2AR to activate either Gαs- or βARR-dependent β2AR signaling pathways. We hypothesized that pepducin-mediated engagement of βARR-dependent β2AR signaling in the heart would be therapeutically advantageous following ischemia/reperfusion (I/R) injury. To test this, wild-type (WT) C57BL/6 mice received three intracardiac injections of either a βARR-biased pepducin (ICL1-9) or a scrambled control pepducin at the time of ischemia (30 min) followed by reperfusion. Assessment of cardiomyocyte death 24h post-I/R using TUNEL staining showed a decrease in cell death in animals treated with ICL1-9 when compared to scrambled pepducin, correlating with decreased infarct size and improved cardiac function as measured by echocardiography. Assessment of cardiac function at later time points showed that the cardioprotective effects of ICL1-9 were preserved over time and resulted in decreased cardiac remodeling. Although βARR1KO mice displayed similar cell death, infarct size, and contractility changes following I/R as their WT counterparts, they did not manifest a cardioprotective response from ICL1-9 treatment, indicating that βARR signaling is essential in relaying ICL1-9-dependent cardioprotection. These results demonstrate that pharmacologically-mediated activation of βARR-biased β2AR signaling provides a therapeutic benefit in the context of I/R-induced injury and may provide an improved strategy for the treatment of acute cardiac injury.

Significance: Heart failure (HF) is a common end point for many underlying cardiovascular diseases. Down-regulation and desensitization of β-adrenergic receptors (β-AR) caused by G-protein-coupled receptor (GPCR) kinase 2 (GRK2) are prominent features of HF. Recent Advances and Critical Issues: Significant progress has been made to understand the pathological role of GRK2 in the heart both as a GPCR kinase and as a molecule that can exert GPCR-independent effects. Inhibition of cardiac GRK2 has proved to be therapeutic in the failing heart and may offer synergistic and additional benefits to β-blocker therapy. However, the mechanisms of how GRK2 directly contributes to the pathogenesis of HF need further investigation, and additional verification of the mechanistic details are needed before GRK2 inhibition can be used for the treatment of HF. Future Directions: The newly identified characteristics of GRK2, including the S-nitrosylation of GRK2 and the localization of GRK2 on mitochondria, merit further investigation. They may contribute to it being a pro-death kinase and result in HF under stressed conditions through regulation of intracellular signaling, including cardiac reduction-oxidation (redox) balance. A thorough understanding of the functions of GRK2 in the heart is necessary in order to finalize it as a candidate for drug development. Antioxid. Redox Signal. 21, 2032–2043.

Background— Liver-X-receptors, LXRα (NR1H3) and LXRβ (NR1H2), encode 2 different but highly homologous isoforms of transcription factors belonging to the nuclear receptor superfamily. Whether LXRα and LXRβ subtypes have discrete roles in the regulation of cardiac physiology/pathology is unknown. We determine the role of each LXR subtype in myocardial ischemia/reperfusion (MI/R) injury. Methods and Results— Mice (wild type; those genetically depleted of LXRα, LXRβ, or both; and those overexpressing LXRα or LXRβ by in vivo intramyocardial adenoviral vector) were subjected to MI/R injury. Both LXRα and LXRβ were detected in wild-type mouse heart. LXRα, but not LXRβ, was significantly upregulated after MI/R. Dual activation of LXRα and LXRβ by natural and synthetic agonists reduced myocardial infarction and improved contractile function after MI/R. Mechanistically, LXR activation inhibited MI/R-induced oxidative stress and nitrative stress, attenuated endoplasmic reticulum stress and mitochondrial dysfunction, and reduced cardiomyocyte apoptosis in ischemic/reperfused myocardium. The aforementioned cardioprotective effects of LXR agonists were impaired in the setting of cardiac-specific gene silencing of LXRα, but not LXRβ subtype. Moreover, LXRα/β double-knockout and LXRα-knockout mice, but not LXRβ-knockout mice, increased MI/R injury, exacerbated MI/R-induced oxidative/nitrative stress, and aggravated endoplasmic reticulum stress and mitochondrial dysfunction. Furthermore, cardiac LXRα, not LXRβ, overexpression via adenoviral transfection suppressed MI/R injury. Conclusions— Our study provides the first direct evidence that the LXRα, but not LXRβ, subtype is a novel endogenous cardiac protective receptor against MI/R injury. Drug development strategies specifically targeting LXRα may be beneficial in treating ischemic heart disease.

BackgroundInjury due to myocardial infarction (MI) is largely irreversible. Once an infarct has occurred, the clinical goal becomes limiting remodeling, preserving left ventricular function, and preventing heart failure. Although traditional approaches (e.g., β-blockers) partially preserve left ventricular function, novel strategies are needed to limit ventricular remodeling post-MI.ObjectivesThe aim of this study was to determine the role of glycogen synthase kinase–3α (GSK-3α) in post-MI remodeling.MethodsMice with cardiomyocyte-specific conditional deletion of Gsk3α and littermate controls underwent sham or MI surgery. Heart function was assessed using serial M-mode echocardiography.ResultsGsk3α deletion in the heart markedly limits remodeling and preserves left ventricular function post-MI. This is due at least in part to dramatic thinning and expansion of the scar in the control hearts, which was less in the heart of knockout (KO) mice. In contrast, the border zone in the KO mice demonstrated a much thicker scar, and there were more viable cardiomyocytes within the scar/border zone. This was associated with less apoptosis and more proliferation of cardiomyocytes in the KO mice. Mechanistically, reduced apoptosis was due, at least in part, to a marked decrease in the Bax/Bcl-2 ratio, and increased cardiomyocyte proliferation was mediated through cyclin E1 and E2F-1 in the hearts of the KO mice.ConclusionsTaken together, these findings show that reducing GSK-3α expression in cardiomyocytes limits ventricular remodeling and preserves cardiac function post-MI. Specifically targeting GSK-3α could be a novel strategy to limit adverse remodeling and heart failure.

Background— Myocardial infarction–induced remodeling includes chamber dilatation, contractile dysfunction, and fibrosis. Of these, fibrosis is the least understood. After myocardial infarction, activated cardiac fibroblasts deposit extracellular matrix. Current therapies to prevent fibrosis are inadequate, and new molecular targets are needed. Methods and Results— Herein we report that glycogen synthase kinase-3β (GSK-3β) is phosphorylated (inhibited) in fibrotic tissues from ischemic human and mouse heart. Using 2 fibroblast-specific GSK-3β knockout mouse models, we show that deletion of GSK-3β in cardiac fibroblasts leads to fibrogenesis, left ventricular dysfunction, and excessive scarring in the ischemic heart. Deletion of GSK-3β induces a profibrotic myofibroblast phenotype in isolated cardiac fibroblasts, in post–myocardial infarction hearts, and in mouse embryonic fibroblasts deleted for GSK-3β. Mechanistically, GSK-3β inhibits profibrotic transforming growth factor-β1/SMAD-3 signaling via interactions with SMAD-3. Moreover, deletion of GSK-3β resulted in the significant increase of SMAD-3 transcriptional activity. This pathway is central to the pathology because a small-molecule inhibitor of SMAD-3 largely prevented fibrosis and limited left ventricular remodeling. Conclusions— These studies support targeting GSK-3β in myocardial fibrotic disorders and establish critical roles of cardiac fibroblasts in remodeling and ventricular dysfunction.

Members of the S100 protein family have been reported to function as endogenous danger signals (alarmins) playing an active role in tissue inflammation and repair when released from necrotic cells. Here, we investigated the role of S100A1, the S100 isoform with highest abundance in cardiomyocytes, when released from damaged cardiomyocytes during myocardial infarction (MI). Patients with acute MI showed significantly increased S100A1 serum levels. Experimental MI in mice induced comparable S100A1 release. S100A1 internalization was observed in cardiac fibroblasts (CFs) adjacent to damaged cardiomyocytes. In vitro analyses revealed exclusive S100A1 endocytosis by CFs, followed by Toll-like receptor 4 (TLR4)-dependent activation of MAP kinases and NF-κB. CFs exposed to S100A1 assumed an immunomodulatory and anti-fibrotic phenotype characterized i.e. by enhanced intercellular adhesion molecule-1 (ICAM1) and decreased collagen levels. In mice, intracardiac S100A1 injection recapitulated these transcriptional changes. Moreover, antibody-mediated neutralization of S100A1 enlarged infarct size and worsened left ventricular functional performance post-MI. Our study demonstrates alarmin properties for S100A1 from necrotic cardiomyocytes. However, the potentially beneficial role of extracellular S100A1 in MI-related inflammation and repair warrants further investigation.

Phospholemman (PLM), when phosphorylated at Ser68, inhibits cardiac Na+/Ca2+exchanger 1 (NCX1) and relieves its inhibition on Na+-K+-ATPase. We have engineered mice in which expression of the phosphomimetic PLM S68E mutant was induced when dietary doxycycline was removed at 5 wk. At 8–10 wk, compared with noninduced or wild-type hearts, S68E expression in induced hearts was ∼35–75% that of endogenous PLM, but protein levels of sarco(endo)plasmic reticulum Ca2+-ATPase, α1- and α2-subunits of Na+-K+-ATPase, α1c-subunit of L-type Ca2+channel, and phosphorylated ryanodine receptor were unchanged. The NCX1 protein level was increased by ∼47% but the NCX1 current was depressed by ∼34% in induced hearts. Isoproterenol had no effect on NCX1 currents but stimulated Na+-K+-ATPase currents equally in induced and noninduced myocytes. At baseline, systolic intracellular Ca2+concentrations ([Ca2+]i), sarcoplasmic reticulum Ca2+contents, and [Ca2+]itransient and contraction amplitudes were similar between induced and noninduced myocytes. Isoproterenol stimulation resulted in much higher systolic [Ca2+]i, sarcoplasmic reticulum Ca2+content, and [Ca2+]itransient and contraction amplitudes in induced myocytes. Echocardiography and in vivo close-chest catheterization demonstrated similar baseline myocardial function, but isoproterenol induced a significantly higher +dP/d t in induced compared with noninduced hearts. In contrast to the 50% mortality observed in mice constitutively overexpressing the S68E mutant, induced mice had similar survival as wild-type and noninduced mice. After ischemia-reperfusion, despite similar areas at risk and left ventricular infarct sizes, induced mice had significantly higher +dP/d t and −dP/d t and lower perioperative mortality compared with noninduced mice. We propose that phosphorylated PLM may be a novel therapeutic target in ischemic heart disease.

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca2+ uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca2+ uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca2+ uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels.

Aims Several adipokines have been proven to improve the therapeutic efficacy of mesenchymal stromal cells (MSCs) when used to treat ischemic heart disease. Asprosin (ASP) is a newly-discovered adipokine. ASP might also predict the severity of coronary pathology. We investigated the role of ASP on MSCs and the effects of ASP-pretreated MSCs on myocardial infarction (MI). Main methods MSCs were labelled with a lentivirus carrying green fluorescent protein (GFP). For in vivo study, after pretreatment with vehicle or ASP, MSCs were injected into infarcted hearts. Cardiac function and fibrosis were then evaluated 4 weeks after the induction of MI and survival of MSCs evaluated after 1 week. MSCs proliferation and migration were investigated after ASP treatment in vitro. MSCs apoptosis induced by hydrogen peroxide (H2O2) was assessed using flow cytometry. Key findings Compared to vehicle-pretreated MSCs, ASP-pretreated MSCs significantly improved the left ventricular ejection fraction (LVEF), and inhibited myocardial fibrosis 4 weeks after MI. ASP pretreatment may have promoted homing of transplanted MSCs. In vitro results showed that ASP had no significant effect on MSC proliferation and migration, but protected these cells from H2O2-induced apoptosis. Among 21 molecules associated with antioxidation and cell death, the antioxidant enzyme SOD2 was significantly upregulated by ASP. Furthermore, ASP treatment inhibited H2O2-induced ROS generation and apoptosis via the activated ERK1/2-SOD2 pathway. Significance This is the first evidence that ASP can regulate MSCs function and enhance MSCs therapy for ischemic heart disease. Furthermore, we demonstrate that ASP protects MSCs from oxidative stress-induced apoptosis via the ERK1/2-SOD2 pathway.

Accumulated pieces of evidence have proved the beneficial effects of melatonin on myocardial ischemia/reperfusion (MI/R) injury, and these effects were largely dependent on melatonin membrane receptor activation. In humans and other mammals, there are two types of melatonin receptors, including the melatonin receptor 1 (MT1, melatonin receptor 1a or MTNR1A) and melatonin receptor 1 (MT2, melatonin receptor 1b or MTNR1B) receptor subtypes. However, which receptor mediates melatonin-conferred cardioprotection remains unclear. In this study, we employed both loss-of-function and gain-of-function approaches to reveal the answer. Mice (wild-type; MT1 or MT2 silencing by in vivo minicircle vector; and those overexpressing MT1 or MT2 by in vivo AAV9 vector) were exposed to MI/R injury. Both MT1 and MT2 were present in wild-type myocardium. MT2, but not MT1, was essentially upregulated after MI/R Melatonin administration significantly reduced myocardial injury and improved cardiac function after MI/R Mechanistically, melatonin treatment suppressed MI/R-initiated myocardial oxidative stress and nitrative stress, alleviated endoplasmic reticulum stress and mitochondrial injury, and inhibited myocardial apoptosis. These beneficial actions of melatonin were absent in MT2-silenced heart, but not the MT1 subtype. Furthermore, AAV9-mediated cardiomyocyte-specific overexpression of MT2, but not MT1, mitigated MI/R injury and improved cardiac dysfunction, which was accompanied by significant amelioration of oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction. Mechanistically, MT2 protected primary cardiomyocytes against hypoxia/reoxygenation injury via MT2/Notch1/Hes1/RORα signaling. Our study presents the first direct evidence that the MT2 subtype, but not MT1, is a novel endogenous cardiac protective receptor against MI/R injury. Medications specifically targeting MT2 may hold promise in fighting ischemic heart disease.

G protein-coupled receptor kinase 5 GRK5 plays a key role in regulating cardiac signaling and its expression is increased in heart failure. GRK5 activity in the nucleus of myocytes has been shown to be detrimental in the setting of pressure-overload hypertrophy. The ubiquitous nuclear transcription factor κB (NF-κB) is involved in the regulation of numerous genes in various tissues, and activation of NF-κB has been shown to be associated with heart disease. Herein, we investigated whether GRK5 can specifically regulate the NF-κB signaling pathway in myocytes. We found that overexpression of GRK5 increased the levels of NF-κB -p50 and p65 in vitro and in vivo, whereas loss of GRK5 resulted in lower cardiac NF-κB levels. Furthermore, increased GRK5 expression induced the phosphorylation status of p65, increased the activity of a NF-κB reporter, and increased NF-κB DNA binding activity in cultured neonatal rat ventricular myocytes. Importantly, siRNA against GRK5 presented with the opposite results in neonatal rat ventricular myocytes as p65 and p50 were decreased, and there was a loss of NF-κB DNA binding activity. The influence of GRK5 on NF-κB expression and activity was dependent on its nuclear localization as overexpression of a mutant GRK5 that cannot enter the nucleus was devoid of NF-κB activation or DNA binding. Our study demonstrates that a novel pathological consequence of GRK5 up-regulation in the injured and failing heart is the induction of NF-κB expression and activity.

In addition to acting on mineralocorticoid receptors, aldosterone has been recently shown to activate the G protein-coupled oestrogen receptor (GPER) in vascular cells. In light of the newly identified role for GPER in vagal cardiac control, we examined whether or not aldosterone activates GPER in rat nucleus ambiguus. Aldosterone produced a dose-dependent increase in cytosolic Ca(2+) concentration in retrogradely labelled cardiac vagal neurons of nucleus ambiguus; the response was abolished by pretreatment with the GPER antagonist G-36, but was not affected by the mineralocorticoid receptor antagonists, spironolactone and eplerenone. In Ca(2+)-free saline, the response to aldosterone was insensitive to blockade of the Ca(2+) release from lysosomes, while it was reduced by blocking the Ca(2+) release via ryanodine receptors and abolished by blocking the IP3 receptors. Aldosterone induced Ca(2+) influx via P/Q-type Ca(2+) channels, but not via L-type and N-type Ca(2+) channels. Aldosterone induced depolarization of cardiac vagal neurons of nucleus ambiguus that was sensitive to antagonism of GPER but not of mineralocorticoid receptor. in vivo studies, using telemetric measurement of heart rate, indicate that microinjection of aldosterone into the nucleus ambiguus produced a dose-dependent bradycardia in conscious, freely moving rats. Aldosterone-induced bradycardia was blocked by the GPER antagonist, but not by the mineralocorticoid receptor antagonists. In summary, we report for the first time that aldosterone decreases heart rate by activating GPER in cardiac vagal neurons of nucleus ambiguus.

The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca2+ influx that was dependent on extracellular Ca2+, was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/d t between WT and KO hearts. At 2–3 days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/d t compared with WT hearts. Compared with WT I/R myocytes, expression of the Na+/Ca2+ exchanger (NCX1) and NCX1 current were increased, expression of the α1-subunit of Na+-K+-ATPase and Na+ pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca2+ concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (SODs) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/R-induced oxidative stress.

Recent clinical observations demonstrate adiponectin (APN), an adipocytokine with potent cardioprotective actions, is significantly reduced following myocardial ischemia/reperfusion (MI/R). However, mechanisms responsible for MI/R-induced hypoadiponectinemia remain incompletely understood. Adult male mice were subjected to 30-min MI followed by varying reperfusion periods. Adipocyte APN mRNA and protein expression and plasma APN and TNFα concentrations were determined. APN expression/production began to decline 3 h after reperfusion (reaching nadir 12 h after reperfusion), returning to control levels 7 days after reperfusion. Plasma TNFα levels began to increase 1 h after reperfusion, peaking at 3 h and returning to control levels 24 h after reperfusion. TNFα knockout significantly increased plasma APN levels 12 h after reperfusion but failed to improve APN expression/production 72 h after reperfusion. In contrast, TNF receptor-1 (TNFR1) knockout significantly restored APN expression 12 and 72 h after reperfusion, suggesting that other TNFR1 binding cytokines contribute to MI/R-induced APN suppression. Among many cytokines increased after MI/R, lymphotoxin-α (LTα) was the only cytokine remaining elevated 24–72 h after reperfusion. LTα knockout did not augment APN levels 12 h post-reperfusion, but did so by 72 h. Finally, in vitro treatment of adipocytes with TNFα and LTα at concentrations seen in MI/R plasma additively inhibited APN expression/production in TNFR1-dependent fashion. Our study demonstrates for the first time that LTα is a novel suppressor of APN expression and contributes to the sustained hypoadiponectinemia following MI/R. Combining anti-TNFα with anti-LTα strategies may achieve the best effects restoring APN in MI/R patients.

Aging is regulated by conserved signaling pathways. The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases regulates several of these pathways, but the role of GSK-3 in aging is unknown. Herein, we demonstrate premature death and acceleration of age-related pathologies in the Gsk3a global KO mouse. KO mice developed cardiac hypertrophy and contractile dysfunction as well as sarcomere disruption and striking sarcopenia in cardiac and skeletal muscle, a classical finding in aging. We also observed severe vacuolar degeneration of myofibers and large tubular aggregates in skeletal muscle, consistent with impaired clearance of insoluble cellular debris. Other organ systems, including gut, liver, and the skeletal system, also demonstrated age-related pathologies. Mechanistically, we found marked activation of mTORC1 and associated suppression of autophagy markers in KO mice. Loss of GSK-3α, either by pharmacologic inhibition or Gsk3a gene deletion, suppressed autophagy in fibroblasts. mTOR inhibition rescued this effect and reversed the established pathologies in the striated muscle of the KO mouse. Thus, GSK-3α is a critical regulator of mTORC1, autophagy, and aging. In its absence, aging/senescence is accelerated in multiple tissues. Strategies to maintain GSK-3α activity and/or inhibit mTOR in the elderly could retard the appearance of age-related pathologies.

Diabetes is a major health problem worldwide. As well-known, diabetes greatly increases cardiac vulnerability to ischemia/reperfusion (I/R) injury, but the underlying mechanisms remain elusive. Nucleostemin (NS) is a nucleolar protein that controls ribosomal biogenesis and exerts cardioprotective effects against I/R injury. However, whether NS-mediated ribosomal biogenesis regulates ischemic vulnerability of diabetic hearts remains unanswered. Utilizing myocardial I/R mouse models, we found that cardiac NS expression significantly increased in response to I/R in normal diet (ND)-fed mice. Surprisingly, cardiac NS failed to be upregulated in high fat diet (HFD)-induced diabetic mice, accompanied by obvious ribosomal dysfunction. Compared with ND group, cardiac specific overexpression of NS by adenovirus (AV) injection significantly restored I/R-induced ribosomal function enhancement, reduced cardiomyocyte apoptosis, improved cardiac function, and decreased infarct sizes in diabetic mice. Notably, co-treatment of homoharringtonine (HHT), a selective inhibitor of ribosomal function, totally blocked NS-mediated cardioprotective effects against I/R injury. Furthermore, in cultured cardiomyocytes, saturated fatty acids treatment, but not high glucose exposure, significantly inhibited simulated I/R-induced NS upregulation and ribosomal function improvement. In conclusion, these data for the first time demonstrate that NS dysregulation induced by saturated fatty acids exposure might be an important cause of increased ischemic vulnerability to I/R injury in diabetic hearts. Targeting NS dysregulation and subsequent ribosomal dysfunction could be a promising therapeutic strategy for diabetic I/R injury management.