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Nrf2, the master gene transcriptor of antioxidant proteins, and SIRT1, the unique Class III histone deacetylase of sirtuins, have been involved in protecting myocardial ischemia/reperfusion (MI/R) injury. However, whether the protective effect of SIRT1 is directly related to the deacetylation of Nrf2 in the pathology of MI/R remains to be investigated. The current study was designed to evaluate the regulation of Nrf2 deacetylation and transcriptional activity by SIRT1 in MI/R. Hypoxia/reoxygenation (H/R) cardiomyocytes and MI/R mice were used to assess the role of SIRT1 in Nrf2 activation. Oxidative stress, cardiac function, LDH release, ROS and infarct size were also evaluated. We found that Nrf2 physically interacted with SIRT1 not only in normal and H/R cardiomyocytes in vitro, but also in Sham or I/R hearts in vivo. Adenovirus induced SIRT1 overexpression resulted in protected H/R induced cell death, accompanied by declined LDH release. Through MI/R in vivo, cardiac overexpression of SIRT1 led to ameliorated cardiac function and infarct size, as well as the decreased cardiac oxidative stress. Notably, such beneficial actions of SIRT1 were blocked by the Nrf2 silence. Mechanically, acetylation of Nrf2 was significantly decreased by SIRT1 overexpression in cardiomyocytes or in whole hearts, which upregulated the downstream signaling pathway of Nrf2. Taken together, we uncovered a clue, for the first time that SIRT1 physically interacts with Nrf2. The cardioprotective effect of SIRT1 overexpression against MI/R is associated with the increased Nrf2 deacetylation and activity. These findings have offered a direct proof and new perspective of post-translational modification in the understanding of oxidative stress and MI/R treatment.

Rationale: Histone LSD1 (lysine-specific demethylase 1) is an important epigenetic antitumor drug target, whose inhibitors are currently in phase I/II clinical trials. However, the potential side effects of LSD1 inhibition in the progress of cardiac remodeling to heart failure remain to be investigated. Objective: To evaluate the roles of myofibroblast- or cardiomyocyte-specific LSD1 deficiency in pressure overload-induced cardiac remodeling. Methods and Results: Adult mouse cardiac fibroblasts, neonatal rat cardiac myocytes, and fibroblasts were isolated, respectively. The myofibroblast-specific and cardiomyocyte-specific LSD1 inducible knockout mice were then generated. We found that LSD1 was increased not only in human dilated cardiomyopathy hearts but also in wild-type mouse heart homogenates and isolated cardiac fibroblasts, following 20 weeks of transverse aortic constriction. The upregulation of LSD1 was also observed in Ang II-treated neonatal rat cardiac fibroblasts, which was reversed by LSD1 silence or its activity inhibition by ORY-1001. These findings suggested a potential involvement of LSD1 in cardiac remodeling. Importantly, myofibroblast-specific LSD1 inducible knockout in vivo significantly alleviated systolic dysfunction, cardiac hypertrophy, and fibrosis, following 6 and 20 weeks of transverse aortic constriction. Mechanistically, through RNA-sequencing and the following Western blot analysis, we found that loss of LSD1 in Ang II-induced myofibroblasts not only inhibited the intracellular upregulation of TGFβ1 (transforming growth factor β1), its downstream effectors Smad2/3 phosphorylation, as well as the phosphorylation of p38, ERK1/2 and JNK, but also reduced the supernatant TGFβ1 secretion, which then decreased myocyte hypertrophy in the indirect coculture model. On the other hand, cardiomyocyte-specific LSD1-inducible knockout in vivo triggered the reprogramming of fetal genes, mild cardiac hypertrophy, and dysfunction under both basal and stressed conditions. Conclusions: Our findings, for the first time, implicate that myofibroblast-specific LSD1 deletion attenuates transverse aortic constriction-induced cardiac remodeling and improves heart function, suggesting that LSD1 is a potential therapeutic target for late-stage heart failure.

Aims Epidermal growth factor receptor (EGFR) is essential to the development of multiple tissues and organs and is a target of cancer therapeutics. Due to the embryonic lethality of global EGFR deletion and conflicting reports of cardiac-overexpressed EGFR mutants, its specific impact on the adult heart, normally or in response to chronic stress, has not been established. Using complimentary genetic strategies to modulate cardiomyocyte-specific EGFR expression, we aim to define its role in the regulation of cardiac function and remodeling. Methods and results A floxed EGFR mouse model with α-myosin heavy chain-Cre-mediated cardiomyocyte-specific EGFR downregulation (CM-EGFR-KD mice) developed contractile dysfunction by 9 weeks of age, marked by impaired diastolic relaxation, as monitored via echocardiographic, hemodynamic and isolated cardiomyocyte contractility analyses. This contractile defect was maintained over time without overt cardiac remodeling until 10 months of age, after which the mice ultimately developed severe heart failure and reduced lifespan. Acute downregulation of EGFR in adult floxed EGFR mice with adeno-associated virus 9 (AAV9)-encoded Cre with a cardiac troponin T promoter (AAV9-cTnT-Cre) recapitulated the CM-EGFR-KD phenotype, while AAV9-cTnT-EGFR treatment of adult CM-EGFR-KD mice rescued the phenotype. Notably, chronic administration of the β-adrenergic receptor (βAR) agonist isoproterenol effectively and reversibly compensated for the contractile dysfunction in the absence of cardiomyocyte hypertrophy in CM-EGFR-KD mice. Mechanistically, EGFR downregulation reduced the expression of protein phosphatase 2 A (PP2A) regulatory subunit Ppp2r3a/PR72, which was associated with decreased phosphorylation of phospholamban (PLB) and Ca2+ clearance, and whose re-expression via AAV9-cTnT-PR72 rescued the CM-EGFR-KD phenotype. Conclusions Altogether our study highlights a previously unrecognized role for EGFR in maintaining contractile homeostasis under physiologic conditions in the adult heart via regulation of PR72 expression. Translational perspective Our study highlights a previously unrecognized role for EGFR in maintaining contractile homeostasis under physiologic conditions in the adult heart via regulation of PR72, a PP2A regulatory subunit with an unknown impact on cardiac function. Further, we have shown that cardiomyocyte-expressed EGFR is required for the promotion of cardiac hypertrophy under conditions of chronic catecholamine stress. Altogether, our study provides new insight into the dynamic nature of cardiomyocyte-specific EGFR.

Background: We previously showed that cardiomyocyte Krϋppel-like factor (KLF) 5 regulates cardiac fatty acid oxidation. As heart failure has been associated with altered fatty acid oxidation, we investigated the role of cardiomyocyte KLF5 in lipid metabolism and pathophysiology of ischemic heart failure. Methods: Using real-time polymerase chain reaction and Western blot, we investigated the KLF5 expression changes in a myocardial infarction (MI) mouse model and heart tissue from patients with ischemic heart failure. Using 2D echocardiography, we evaluated the effect of KLF5 inhibition after MI using pharmacological KLF5 inhibitor ML264 and mice with cardiomyocyte-specific KLF5 deletion (αMHC [α-myosin heavy chain]-KLF5 −/ − ). We identified the involvement of KLF5 in regulating lipid metabolism and ceramide accumulation after MI using liquid chromatography–tandem mass spectrometry, and Western blot and real-time polymerase chain reaction analysis of ceramide metabolism–related genes. We lastly evaluated the effect of cardiomyocyte-specific KLF5 overexpression (αMHC-rtTA [reverse tetracycline-controlled transactivator]-KLF5) on cardiac function and ceramide metabolism, and rescued the phenotype using myriocin to inhibit ceramide biosynthesis. Results: KLF5 mRNA and protein levels were higher in human ischemic heart failure samples and in rodent models at 24 hours, 2 weeks, and 4 weeks post–permanent left coronary artery ligation. αMHC-KLF5 −/− mice and mice treated with ML264 had higher ejection fraction and lower ventricular volume and heart weight after MI. Lipidomic analysis showed that αMHC-KLF5 −/ − mice with MI had lower myocardial ceramide levels compared with littermate control mice with MI, although basal ceramide content of αMHC-KLF5 −/ − mice was not different in control mice. KLF5 ablation suppressed the expression of SPTLC1 and SPTLC2 (serine palmitoyltransferase [SPT] long-chain base subunit ()1 2, respectively), which regulate de novo ceramide biosynthesis. We confirmed our previous findings that myocardial SPTLC1 and SPTLC2 levels are increased in heart failure patients. Consistently, αMHC-rtTA-KLF5 mice showed increased SPTLC1 and SPTLC2 expression, higher myocardial ceramide levels, and systolic dysfunction beginning 2 weeks after KLF5 induction. Treatment of αMHC-rtTA-KLF5 mice with myriocin that inhibits SPT, suppressed myocardial ceramide levels and alleviated systolic dysfunction. Conclusions: KLF5 is induced during the development of ischemic heart failure in humans and mice and stimulates ceramide biosynthesis. Genetic or pharmacological inhibition of KLF5 in mice with MI prevents ceramide accumulation, alleviates eccentric remodeling, and increases ejection fraction. Thus, KLF5 emerges as a novel therapeutic target for the treatment of ischemic heart failure.

Objective Acute myocardial infarction (AMI) initiates pathological inflammation which aggravates tissue damage and causes heart failure. Lysophosphatidic acid (LPA), produced by autotaxin (ATX), promotes inflammation and the development of atherosclerosis. The role of ATX/LPA signaling nexus in cardiac inflammation and resulting adverse cardiac remodeling is poorly understood. Approach and results We assessed autotaxin activity and LPA levels in relation to cardiac and systemic inflammation in AMI patients and C57BL/6 (WT) mice. Human and murine peripheral blood and cardiac tissue samples showed elevated levels of ATX activity, LPA, and inflammatory cells following AMI and there was strong correlation between LPA levels and circulating inflammatory cells. In a gain of function model, lipid phosphate phosphatase-3 (LPP3) specific inducible knock out (Mx1-Plpp3Δ) showed higher systemic and cardiac inflammation after AMI compared to littermate controls (Mx1-Plpp3fl/fl); and a corresponding increase in bone marrow progenitor cell count and proliferation. Moreover, in Mx1- Plpp3Δ mice, cardiac functional recovery was reduced with corresponding increases in adverse cardiac remodeling and scar size (as assessed by echocardiography and Masson's Trichrome staining). To examine the effect of ATX/LPA nexus inhibition, we treated WT mice with the specific pharmacological inhibitor, PF8380, twice a day for 7 days post AMI. Inhibition of the ATX/LPA signaling nexus resulted in significant reduction in post-AMI inflammatory response, leading to favorable cardiac functional recovery, reduced scar size and enhanced angiogenesis. Conclusion ATX/LPA signaling nexus plays an important role in modulating inflammation after AMI and targeting this mechanism represents a novel therapeutic target for patients presenting with acute myocardial injury.

Objective Post myocardial infarction (MI) fibrosis has been identified as an important factor in the progression of heart failure. Previous studies have revealed that microRNA-21 (miR-21) plays an important role in the pathogenesis of fibrosis. The purpose of this study was to explore the role of miR-21 in post-MI cardiac fibrosis. Material and methods MI was established in wild-type (WT) and miR-21 knockout (KO) mice. Primary mice cardiac fibroblasts (CFs) were isolated from WT and miR-21 KO mice and were treated with angiotensin II (Ang II) or Sprouty1 (Spry1) siRNA. Histological analysis and echocardiography were used to determine the extent of fibrosis and cardiac function. Results Compared with WT mice, miR-21 KO mice displayed smaller fibrotic areas and decreased expression of fibrotic markers and inflammatory cytokines. In parallel, Ang II-induced myofibroblasts transformation was partially inhibited upon miR-21 KO in primary CFs. Mechanistically, we found that the expression of Spry1, a previously reported target of miR-21, was markedly increased in miR-21 KO mice post MI, further inhibiting ERK1/2 activation. In vitro studies showed that Ang II activated ERK1/2/TGF-β/Smad2/3 pathway. Phosphorylated Smad2/3 further enhanced the expression of α-SMA and FAP and may promote the maturation of miR-21, thereby downregulating Spry1. Additionally, these effects of miR-21 KO on fibrosis were reversed by siRNA-mediated knockdown of Spry1. Conclusion Our findings suggest that miR-21 promotes post-MI fibrosis by targeting Spry1. Furthermore, it mediates a positive feedback on Ang II, thereby inducing the ERK/TGF-β/Smad pathway. Therefore, targeting the miR-21-Spry1 axis may be a promising therapeutic option for ameliorating post-MI cardiac fibrosis.

Background: Cardiac hypertrophic growth is mediated by robust changes in gene expression and changes that underlie the increase in cardiomyocyte size. The former is regulated by RNA polymerase II (pol II) de novo recruitment or loss; the latter involves incremental increases in the transcriptional elongation activity of pol II that is preassembled at the transcription start site. The differential regulation of these distinct processes by transcription factors remains unknown. Forkhead box protein O1 (FoxO1) is an insulin-sensitive transcription factor that is also regulated by hypertrophic stimuli in the heart. However, the scope of its gene regulation remains unexplored. Methods: To address this, we performed FoxO1 chromatin immunoprecipitation–deep sequencing in mouse hearts after 7 days of isoproterenol injections (3 mg·kg −1 ·mg −1 ), transverse aortic constriction, or vehicle injection/sham surgery. Results: Our data demonstrate increases in FoxO1 chromatin binding during cardiac hypertrophic growth, which positively correlate with extent of hypertrophy. To assess the role of FoxO1 on pol II dynamics and gene expression, the FoxO1 chromatin immunoprecipitation–deep sequencing results were aligned with those of pol II chromatin immunoprecipitation–deep sequencing across the chromosomal coordinates of sham- or transverse aortic constriction–operated mouse hearts. This uncovered that FoxO1 binds to the promoters of 60% of cardiac-expressed genes at baseline and 91% after transverse aortic constriction. FoxO1 binding is increased in genes regulated by pol II de novo recruitment, loss, or pause-release. In vitro, endothelin-1– and, in vivo, pressure overload–induced cardiomyocyte hypertrophic growth is prevented with FoxO1 knockdown or deletion, which was accompanied by reductions in inducible genes, including Comtd1 in vitro and Fstl1 and Uck2 in vivo. Conclusions: Together, our data suggest that FoxO1 may mediate cardiac hypertrophic growth via regulation of pol II de novo recruitment and pause-release; the latter represents the majority (59%) of FoxO1-bound, pol II–regulated genes after pressure overload. These findings demonstrate the breadth of transcriptional regulation by FoxO1 during cardiac hypertrophy, information that is essential for its therapeutic targeting.

Background: Diabetes mellitus exacerbates myocardial ischemia/reperfusion (MI/R) injury by incompletely understood mechanisms. Adipocyte dysfunction contributes to remote organ injury. However, the molecular mechanisms linking dysfunctional adipocytes to increased MI/R injury remain unidentified. The current study attempted to clarify whether and how small extracellular vesicles (sEV) may mediate pathological communication between diabetic adipocytes and cardiomyocytes, exacerbating MI/R injury. Methods: Adult male mice were fed a normal or a high-fat diet for 12 weeks. sEV (from diabetic serum, diabetic adipocytes, or high glucose/high lipid-challenged nondiabetic adipocytes) were injected intramyocardially distal of coronary ligation. Animals were subjected to MI/R 48 hours after injection. Results: Intramyocardial injection of diabetic serum sEV in the nondiabetic heart significantly exacerbated MI/R injury, as evidenced by poorer cardiac function recovery, larger infarct size, and greater cardiomyocyte apoptosis. Similarly, intramyocardial or systemic administration of diabetic adipocyte sEV or high glucose/high lipid-challenged nondiabetic adipocyte sEV significantly exacerbated MI/R injury. Diabetic epididymal fat transplantation significantly increased MI/R injury in nondiabetic mice, whereas administration of a sEV biogenesis inhibitor significantly mitigated MI/R injury in diabetic mice. A mechanistic investigation identified that miR-130b-3p is a common molecule significantly increased in diabetic serum sEV, diabetic adipocyte sEV, and high glucose/high lipid-challenged nondiabetic adipocyte sEV. Mature (but not primary) miR-130b-3p was significantly increased in the diabetic and nondiabetic heart subjected to diabetic sEV injection. Whereas intramyocardial injection of a miR-130b-3p mimic significantly exacerbated MI/R injury in nondiabetic mice, miR-130b-3p inhibitors significantly attenuated MI/R injury in diabetic mice. Molecular studies identified AMPKα1/α2, Birc6, and Ucp3 as direct downstream targets of miR-130b-3p. Overexpression of these molecules (particularly AMPKα2) reversed miR-130b-3p induced proapoptotic/cardiac harmful effect. Finally, miR-130b-3p levels were significantly increased in plasma sEV from patients with type 2 diabetes mellitus. Incubation of cardiomyocytes with diabetic patient sEV significantly exacerbated ischemic injury, an effect blocked by miR-130b-3p inhibitor. Conclusions: We demonstrate for the first time that miR-130b-3p enrichment in dysfunctional adipocyte-derived sEV, and its suppression of multiple antiapoptotic/cardioprotective molecules in cardiomyocytes, is a novel mechanism exacerbating MI/R injury in the diabetic heart. Targeting miR-130b-3p mediated pathological communication between dysfunctional adipocytes and cardiomyocytes may be a novel strategy attenuating diabetic exacerbation of MI/R injury.

INTRODUCTION: Acute myocardial infarction (AMI) and resulting cardiac damage and heart failure are leading causes of morbidity and mortality worldwide. Multiple studies have examined the utility of CD34(+) cells for the treatment of acute and ischemic heart disease. However, the optimal strategy to enrich CD34 cells from clinical sources is not known. We examined the efficacy of fluorescence activated cell sorting (FACS) and magnetic beads cell sorting (MACS) methods for CD34 cell isolation from mobilized human mononuclear peripheral blood cells (mhPBMNCs). METHODS: mhPBCs were processed following acquisition using FACS or MACS according to clinically established protocols. Cell viability, CD34 cell purity and characterization of surface marker expression was assessed using a flow cytometer. For in vivo characterization of cardiac repair, we conducted LAD ligation surgery on 8–10 weeks female NOD/SCID mice followed by intramyocardial transplantation of unselected mhPBMNCs, FACS or MACS enriched CD34(+) cells. RESULTS: Both MACS and FACS isolation methods achieved high purity rates, viability, and enrichment of CD34(+) cells. In vivo studies following myocardial infarction demonstrated retention of CD34(+) in the peri-infarct region for up to 30 days after transplantation. Retained CD34(+) cells were associated with enhanced angiogenesis and reduced inflammation compared to unselected mhPBMNCs or PBS treatment arms. Cardiac scar and fibrosis as assessed by immunohistochemistry were reduced in FACS and MACS CD34(+) treatment groups. Finally, reduced scar and augmented angiogenesis resulted in improved cardiac functional recovery, both on the global and regional function and remodeling assessments by echocardiography. CONCLUSION: Cell based therapy using enriched CD34(+) cells sorted by FACS or MACS result in better cardiac recovery after ischemic injury compared to unselected mhPBMNCs. Both enrichment techniques offer excellent recovery and purity and can be equally used for clinical applications.

Sodium (±)‐5‐bromo‐2‐(a‐hydroxypentyl) benzoate (generic name: brozopine, BZP) has been reported to protect against stroke‐induced brain injury and was approved for Phase II clinical trials for treatment of stroke‐related brain damage by the China Food and Drug Administration (CFDA). However, the role of BZP in cardiac diseases, especially in pressure overload‐induced cardiac hypertrophy and heart failure, remains to be investigated. In the present study, angiotensin II stimulation and transverse aortic constriction were employed to induce cardiomyocyte hypertrophy in vitro and in vivo, respectively, prior to the assessment of myocardial cell autophagy. We observed that BZP administration ameliorated cardiomyocyte hypertrophy and excessive autophagic activity. Further results indicated that AMP‐activated protein kinase (AMPK)‐mediated activation of the mammalian target of rapamycin (mTOR) pathway likely played a role in regulation of autophagy by BZP after Ang II stimulation. The activation of AMPK with metformin reversed the BZP‐induced suppression of autophagy. Finally, for the first time, we demonstrated that BZP could protect the heart from pressure overload‐induced hypertrophy and dysfunction, and this effect is associated with its inhibition of maladaptive cardiomyocyte autophagy through the AMPK‐mTOR signalling pathway. These findings indicated that BZP may serve as a promising compound for treatment of pressure overload‐induced cardiac remodelling and heart failure.

Aims Estrogen plays an important role in cardioprotection. Animal experiments showed that the G-protein coupled estrogen receptor 30 (GPR30) specific agonist G1 could reduce post-ischemic dysfunction and inhibit cardiac fibroblast proliferation. However, the underlying mechanism of action is not clear. The current study tests the hypothesis that GPR30 reduces myocardial infarct area and fibrosis in female ovariectomized (OVX) mice by activating the PI3K/AKT pathway. Main methods In this study, we established a myocardial infarction (MI) animal model derived from OVX C57BL/6 female mice, and investigated the effect of G1 on cardiac function by echocardiography and Hemodynamics, morphology and expression of fibrosis-related and apoptosis-related proteins by Masson's trichrome and H&E, Immunofluorescence, Western blotting and TUNEL. Key findings Combination with OVX significantly increased myocardial fibrosis and MI area compared to MI treatment alone, as determined by echocardiography and hemodynamics. Further addition of G1 changed the expression of apoptosis-related proteins, decreased the levels of tumor necrosis factor-α and interleukin-10, and reduced the degree of myocardial fibrosis and myocardial infarct area. Primary cultured cardiac fibroblasts (CFs) were subjected to hypoxia/serum deprivation (H/SD) simulating the in vivo ischemia model. When the PI3K/AKT pathway was inhibited by wortmanin in H/SD CFs, G1 failed to induce significant changes in the expression of apoptosis-related proteins. Significance It suggested that GPR30 may improve cardiac function in female OVX mice by activating the PI3K/AKT pathway and reducing myocardial infarct size and fibrosis.

Myocardial ischemia/reperfusion injury is a common clinical problem and can result in severe cardiac dysfunction. Previous studies have demonstrated the protection of electroacupuncture against myocardial ischemia/reperfusion injury. However, the role of X-box binding protein I (XBP1) signaling pathway in the protection of electroacupuncture was still elusive. Thus, we designed this study and demonstrated that electroacupuncture significantly improved cardiac function during myocardial ischemia/reperfusion injury and reduced cardiac infarct size. Electroacupuncture treatment further inhibited cardiac injury manifested by the decrease of the activities of serum lactate dehydrogenase and creatine kinase-MB. The results also revealed that electroacupuncture elevated the expressions of XBP1, glucose-regulated protein 78 (GRP78), Akt, and Bcl-2 and decreased the Bax and cleaved Caspase 3 expressions. By using the inhibitor of XBP1 in vitro, the results revealed that suppression of XBP1 expression could markedly increase the activities of lactate dehydrogenase and creatine kinase-MB and cell apoptosis, thus exacerbating stimulated ischemia/reperfusion-induced H9c2 cell injury. Compared with stimulated ischemia/reperfusion group, inhibition of XBP1 inhibited the downstream GRP78 and Akt expressions during stimulated ischemia/reperfusion injury. Collectively, our data demonstrated that electroacupuncture treatment activated XBP1/GRP78/Akt signaling to protect hearts from myocardial ischemia/reperfusion injury. These findings revealed the underlying mechanisms of electroacupuncture protection against myocardial ischemia/reperfusion injury and may provide novel therapeutic targets for the clinical treatment of myocardial ischemia/reperfusion injury.

Background Diabetic cardiomyopathy (DCM) is characterized by cardiac dysfunction and cardiomyocyte injury, which induced by metabolic disorder. Nowadays, there is still a lack of drugs for the treatment of DCM. Growth differentiation factor 11 (GDF11) is a novel member of the transforming growth factor β superfamily that alleviates cardiac hypertrophy, myocardial infarction, and vascular injury by regulating oxidative stress, inflammation, and cell survival. However, the roles and underlying mechanisms of GDF11 in DCM remain largely unknown. Methods In this study, we sought to determine whether GDF11 could prevent DCM. The mouse model of diabetes was established by administering a high-fat diet and intraperitoneal injecting streptozotocin. After that, intramyocardial injection of an adeno-associated virus carrying GDF11 gene was used to achieve myocardium-specific overexpression. Results Our data showed that GDF11 overexpression remarkably improved cardiac dysfunction and interstitial fibrosis by reducing the levels of reactive oxygen species and protecting against cardiomyocyte loss. Mechanistically, decreased sirtuin 1 (SIRT1) expression was observed in diabetic mice, which was significantly increased after GDF11 overexpression. To further explore how SIRT1 mediates the role of GDF11, the selective inhibitor EX527 was used to block SIRT1 signaling pathway, which abolished the protective effects of GDF11 against DCM. In vitro studies confirmed that GDF11 protected against H9c2 cell injury in high glucose and palmitate by attenuating oxidative injury and apoptosis, and these effects were also eliminated by SIRT1 depletion. Conclusion Our results demonstrated for the first time that GDF11 protected against DCM by regulating SIRT1 signaling pathway, and GDF11 had the potential to become a novel target for reversing cardiac dysfunction in diabetic patients.

A growing body of evidence shows that altering the inflammatory response by alternative macrophage polarization is protective against complications related to acute myocardial infarction (MI). We have previously shown that oral azithromycin (AZM), initiated prior to MI, reduces inflammation and its negative sequelae on the myocardium. Here, we investigated the immunomodulatory role of a liposomal AZM formulation (L-AZM) in a clinically relevant model to enhance its therapeutic potency and avoid off-target effects. L-AZM (40 or 10 mg/kg, IV) was administered immediately post-MI and compared to free AZM (F-AZM). L-AZM reduced cardiac toxicity and associated mortality by 50% in mice. We observed a significant shift favoring reparatory/anti-inflammatory macrophages with L-AZM formulation. L-AZM use resulted in a remarkable decrease in cardiac inflammatory neutrophils and the infiltration of inflammatory monocytes. Immune cell modulation was associated with the downregulation of pro-inflammatory genes and the upregulation of anti-inflammatory genes. The immunomodulatory effects of L-AZM were associated with a reduction in cardiac cell death and scar size as well as enhanced angiogenesis. Overall, L-AZM use enhanced cardiac recovery and survival after MI. Importantly, L-AZM was protective from F-AZM cardiac off-target effects. We demonstrate that the liposomal formulation of AZM enhances the drug's efficacy and safety in an animal model of acute myocardial injury. This is the first study to establish the immunomodulatory properties of liposomal AZM formulations. Our findings strongly support clinical trials using L-AZM as a novel and clinically relevant therapeutic target to improve cardiac recovery and reduce heart failure post-MI in humans.

Recent data supporting any benefit of stem cell therapy for ischemic heart disease have suggested paracrine-based mechanisms via extracellular vesicles (EVs) including exosomes. We have previously engineered cardiac-derived progenitor cells (CDCs) to express a peptide inhibitor, βARKct, of G protein-coupled receptor kinase 2, leading to improvements in cell proliferation, survival, and metabolism. In this study, we tested whether βARKct-CDC EVs would be efficacious when applied to stressed myocytes in vitro and in vivo. When isolated EVs from βARKct-CDCs and control GFP-CDCs were added to cardiomyocytes in culture, they both protected against hypoxia-induced apoptosis. We tested whether these EVs could protect the mouse heart in vivo, following exposure either to myocardial infarction (MI) or acute catecholamine toxicity. Both types of EVs significantly protected against ischemic injury and improved cardiac function after MI compared with mice treated with EVs from mouse embryonic fibroblasts; however, βARKct EVs treated mice did display some unique beneficial properties including significantly altered pro- and anti-inflammatory cytokines. Importantly, in a catecholamine toxicity model of heart failure (HF), myocardial injections of βARKct-containing EVs were superior at preventing HF compared with control EVs, and this catecholamine toxicity protection was recapitulated in vitro. Therefore, introduction of the βARKct into cellular EVs can have improved reparative properties in the heart especially against catecholamine damage, which is significant as sympathetic nervous system activity is increased in HF. NEW & NOTEWORTHY βARKct, the peptide inhibitor of GRK2, improves survival and metabolic functions of cardiac-derived progenitor cells. As any benefit of stem cells in the ischemic and injured heart suggests paracrine mechanisms via secreted EVs, we investigated whether CDC-βARKct engineered EVs would show any benefit over control CDC-EVs. Compared with control EVs, βARKct-containing EVs displayed some unique beneficial properties that may be due to altered pro- and anti-inflammatory cytokines within the vesicles.

Significance Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. G protein-coupled receptor (GPCR) kinase 5 (GRK5) has been shown to cause deleterious effects on the cardiomyocyte during HF; however, its effects in cardiac fibroblasts, the crucial cell type responsible for maintaining the structural integrity of the heart, is not understood. Here, we use in vitro and in vivo methods to demonstrate that inhibition of GRK5 inhibits fibroblast activation and attenuates the fibrotic response in the heart., Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor kinase 5 (GRK5) is an important mediator of cardiovascular homeostasis through dampening of GPCR signaling, and is expressed in the heart and up-regulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium-calmodulin (Ca2+-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of nuclear factor of activated T cells (NFAT). Interestingly, NFAT is also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts; however, its pathophysiological role in these crucial cardiac cells is unknown. We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits angiotensin II (AngII)-mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice led to decreased fibrosis and cardiac hypertrophy after chronic AngII infusion or after ischemic injury compared to nontransgenic littermate controls (NLCs). Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation. These data demonstrate that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrate the potential beneficial effects of GRK5 inhibition in the context of cardiac disease.

Introduction and Hypothesis: Acute myocardial infarction (AMI) is the leading cause of morbidity and mortality worldwide. The innate immune response plays a major role in cardiac remodeling after AMI. Lysophosphatidic acid (LPA), produced by autotaxin (ATX) and degraded by Lipid phosphate phosphatase 3 (LPP3), regulates monocytosis and promotes inflammation. However, the role of LPP3 in post-AMI inflammation is not understood. Here, we investigated the possible role of Myeloid-specific Plpp3 KO mice in cardiac and systemic inflammation and resulting in adverse cardiac remodeling post-MI. Methods and Results: To generate mice with Myeloid-specific Plpp3 deletion , female Plpp3 fl/fl mice were crossed to male Plpp3 mice expressing Cre recombinase under the control of the LysM promoter to generate lysm-plpp3. mice. These mice and their littermate control underwent MI or sham surgery. Inflammatory cell content was assessed using flow cytometry and immunohistochemistry. Cardiac function and scar size were assessed by echocardiography and Mason Trichrome staining, respectively. Increased number of Ly6C hi monocytes (CD45 + /Ly6C/G hi /CD115 hi ) and pro-inflammatory macrophages (CD45 + /F4-80 + /CD11b + ) (CD45 + /F4-80 + /CD86 + ), in cardiac tissue of Cre+LysM mice was observed compared to Cre-fl/fl littermate controls during peak post-MI inflammation, as assessed by flow cytometry ( Fig.1A-C ). This increase in inflammatory cells and inflammation may be the consequence of the significant increase in bone marrow and spleen progenitor cell count and proliferation. Moreover, Cre+LysM mice cardiac functional recovery was also reduced significantly ( Fig. 1D-F ) as assessed by echocardiography. Conclusion: Myeloid-specific Plpp3 deletion increases the deleterious effects of inflammation on the ischemic myocardium and ATX/ LPA signaling could represent a novel therapeutic target for future clinical studies of coronary heart diseases.

A growing body of evidence shows that altering the inflammatory response by alternative macrophage polarization is protective against complications related to acute myocardial infarction (MI). We have previously shown that oral azithromycin (AZM), initiated prior to MI, reduces inflammation and its negative sequelae on the myocardium. Here, we investigated the immunomodulatory role of a liposomal AZM formulation (L-AZM) in a clinically relevant model to enhance its therapeutic potency and avoid off-target effects. L-AZM (40 or 10 mg/kg, IV) was administered immediately post-MI and compared to free AZM (F-AZM). L-AZM reduced cardiac toxicity and associated mortality by 50% in mice. We observed a significant shift favoring reparatory/anti-inflammatory macrophages with L-AZM formulation. L-AZM use resulted in a remarkable decrease in cardiac inflammatory neutrophils and the infiltration of inflammatory monocytes. Immune cell modulation was associated with the downregulation of pro-inflammatory genes and the upregulation of anti-inflammatory genes. The immunomodulatory effects of L-AZM were associated with a reduction in cardiac cell death and scar size as well as enhanced angiogenesis. Overall, L-AZM use enhanced cardiac recovery and survival after MI. Importantly, L-AZM was protective from F-AZM cardiac off-target effects. We demonstrate that the liposomal formulation of AZM enhances the drug’s efficacy and safety in an animal model of acute myocardial injury. This is the first study to establish the immunomodulatory properties of liposomal AZM formulations. Our findings strongly support clinical trials using L-AZM as a novel and clinically relevant therapeutic target to improve cardiac recovery and reduce heart failure post-MI in humans.

Complex tissue regeneration is extremely rare among adult mammals. An exception, however, is the superior tissue healing of multiple organs in spiny mice (Acomys). While Acomys species exhibit the remarkable ability to heal complex tissue with minimal scarring, little is known about their cardiac structure and response to cardiac injury. In this study, we first examined baseline Acomys cardiac anatomy and function in comparison with commonly used inbred and outbred laboratory Mus strains (C57BL6 and CFW). While our results demonstrated comparable cardiac anatomy and function between Acomys and Mus, Acomys exhibited a higher percentage of cardiomyocytes displaying distinct characteristics. In response to myocardial infarction, all animals experienced a comparable level of initial cardiac damage. However, Acomys demonstrated superior ischemic tolerance and cytoprotection in response to injury as evidenced by cardiac functional stabilization, higher survival rate, and smaller scar size 50 days after injury compared to the inbred and outbred mouse strains. This phenomenon correlated with enhanced endothelial cell proliferation, increased angiogenesis, and medium vessel maturation in the peri-infarct and infarct regions. Overall, these findings demonstrate augmented myocardial preservation in spiny mice post-MI and establish Acomys as a new adult mammalian model for cardiac research.

Background Myocardial ischemia/reperfusion (MI/R) injury imposes devastating cardiovascular sequelae in particular cardiac dysfunction as a result of restored blood flow. However, the mechanism behind MI/R injury remains elusive. Mitochondrial ubiquitin ligase (MITOL/MARCH5) is localized at the mitochondria‐ER contact site and may be activated in response to a variety of pathophysiological processes, such as apoptosis, mitochondrial injury, ER stress, hypoxia, and reactive oxygen species (ROS) generation. Irisin as a cleaved product of fibronectin type III domain‐containing protein 5 (FNDC5) displays cardioprotection in diverse cardiac diseases. Methods This study was designed to examine the role of irisin and MITOL in MI/R injury. Male C57BL/6J mice (8‐10‐week‐old) were administered adenovirus MITOL shRNA through intracardiac injection followed by MI/R surgery through ligation and release the slipknot of cardiac left anterior descending coronary artery. Results Our results showed that irisin improved myocardial function in the face of MI/R injury as evidenced by reduced myocardial infarct size, apoptotic rate, serum lactate dehydrogenase (LDH), ROS generation, and malondialdehyde (MDA) levels as well as lessened ER stress injury. Moreover, our results indicated that protective role of irisin was mediated by upregulation of MITOL. Irisin also protected H9c2 cells against simulated I/R through negating ER stress, apoptosis, ROS and MDA levels, as well as facilitating superoxide dismutase (SOD) by way of elevated MITOL expression. Conclusions To this end, our data favored that irisin pretreatment protects against MI/R injury, ER stress, ROS production, and mitochondrial homeostasis through upregulation of MITOL. These findings depicted the therapeutic potential of irisin and MITOL in the management of MI/R injury in patients with ST‐segment elevation., Irisin improves cardiac function against myocardial ischemia/reperfusion (MI/R) injury, decreases apoptosis and myocardial infarct size following MI/R injury, alleviates reactive oxygen species (ROS) production following MI/R injury, and protects against MI/R injury, ER stress, and mitochondrial homeostasis through upregulation of MITOL

BackgroundExosomes derived from adipose-derived mesenchymal stem cells can potentially protect cardiomyocytes from myocardial ischemia reperfusion injury. It's notable that exosomes derived from adipose-derived mesenchymal stem cells underwent anoxia preconditioning showed a better cardioprotective effect than that without anoxia. Here, in vitro and in vivo studies were used to investigate the cardioprotective effects against myocardial ischemia reperfusion injury of exosomes derived from adipose-derived mesenchymal stem cells with (Int-EXO) or without anoxia (NC-EXO), respectively. Methods: Adipose-derived mesenchymal stem cells and exosomes were identified by western blot, flow cytometry, transmission electron microscopy, and nanosight. An exosome tracer assay identified exosomes absorbed by cells. An in vitro model using mice cardiomyocytes for studying anoxia-reoxygenation and an in vivo mice model of MIRI were used to investigate the cardioprotective effects of NC-Exo and Int-Exo, respectively.ResultsWe discovered that treatment with NC-EXO and Int-EXO significantly reduced the infarct size and attenuated cardiomyocyte apoptosis, In addition, Int-EXO group had a less infarct size and apoptosis degree. The mechanism revealed by RNA sequencing showed that 40 miRNAs were up-regulated in Int-EXO compared to NC-EXO. 10 of these miRNAs could bind thioredoxin-interacting protein as a downstream target gene; among these, the top-discrepant miRNA224-5p was selected for further study. Dual luciferase reporter assay and rescue study verified TXNIP as a target gene for miR-224-5p. Furthermore, the cellular death signaling pathway which Int-EXO involved in mediating was in a special form of apoptosis, not pyroptosis, induced by activated thioredoxin-interacting protein-pyroptosis-caspase1 pathway in gasdermin D-deficient cells. ConclusionThe research demonstrated adipose-derived mesenchymal stem cells exosomes attenuated MIRI by inhibiting pyroptosis-induced apoptosis in cardiomyocytes which are lack of gasdermin D. The cardioprotective effect of Int-EXO was more significant than that of NC-EXO, possibly due to treated with anoxia preconditioning, adipose-derived mesenchymal stem cells product more miRNAs targeting thioredoxin-interacting protein in exosomes to alleviate pyroptosis-induced apoptosis. These findings provide new insights into the pathogenesis and methods for intervention of myocardial ischemia reperfusion injury.

Cardiac contractility is regulated by the intracellular Ca 2+ concentration fluxes which are actively regulated by multiple channels and transporters. Ca 2+ uptake into the mitochondrial matrix is precisely controlled by the highly Ca 2+ selective channel, Mitochondrial Calcium Uniporter (MCU). Earlier studies on the cardiac-specific acute MCU knockout and a transgenic dominant-negative MCU mice have demonstrated that mitochondrial Ca 2+ ( m Ca 2+ ) signaling is necessary for cardiac ‘‘fight-or-flight’’ contractile response, however, the role of m Ca 2+ buffering to shape global cytosolic Ca 2+ levels and affect E-C coupling, particularly the Ca 2+ transient, on a beat-to-beat basis still remains to be solved. Our earlier studies have demonstrated that loss of MCU Regulator 1 (MCUR1) in cardiomyocytes results in the impaired m Ca 2+ uptake. We have now employed the cardiac-specific MCUR1 knockout mouse to dissect the precise role of MCU in regulating cytosolic Ca 2+ transients associated with excitation-contraction (E-C) coupling and cardiac function. Results from our studies including the in vivo analyses of cardiac physiology during normal and pressure-overloaded mouse models and in vitro experiments including single-cell cardiac contractility, calcium transients, and electrophysiology measurements demonstrate that MCUR1/MCU regulated m Ca 2+ buffering in cardiomyocytes, although insignificant under basal condition, becomes critical in stress induced conditions and actively participates in regulating the c Ca 2+ transients. Also, the ablation of MCUR1 in cardiomyocytes during stress conditions prevents m Ca 2+ overload and subsequent mROS overproduction. Our data indicate that MCUR1 ablation offers protection against pressure-overload cardiac hypertrophy. In summary, our results provide critical insights into the mechanisms by which the MCU channel contributes in regulating the contractile function of the cardiomyocytes and the role of m Ca 2+ in the development and progression of heart failure.

Aims Myocardial infarction (MI) is the most common cause of heart failure (HF) worldwide. G protein-coupled receptor kinase 5 (GRK5) is upregulated in failing human myocardium and promotes maladaptive cardiac hypertrophy in animal models. However, the role of GRK5 in ischemic heart disease is still unknown. In this study, we evaluated whether myocardial GRK5 plays a critical role post-MI in mice and included the examination of specific cardiac immune and inflammatory responses. Methods and results Cardiomyocyte-specific GRK5 overexpressing transgenic mice (TgGRK5) and non-transgenic littermate control (NLC) mice as well as cardiomyocyte-specific GRK5 knockout mice (GRK5cKO) and wild type (WT) were subjected to MI and, functional as well as structural changes together with outcomes were studied. TgGRK5 post-MI mice showed decreased cardiac function, augmented left ventricular dimension and decreased survival rate compared to NLC post-MI mice. Cardiac hypertrophy and fibrosis as well as fetal gene expression were increased post-MI in TgGRK5 compared to NLC mice. In TgGRK5 mice, GRK5 elevation produced immuno-regulators that contributed to the elevated and long-lasting leukocyte recruitment into the injured heart and ultimately to chronic cardiac inflammation. We found an increased presence of pro-inflammatory neutrophils and macrophages as well as neutrophils, macrophages and T-lymphocytes at 4-days and 8-weeks respectively post-MI in TgGRK5 hearts. Conversely, GRK5cKO mice were protected from ischemic injury and showed reduced early immune cell recruitment (predominantly monocytes) to the heart, improved contractility and reduced mortality compared to WT post-MI mice. Interestingly, cardiomyocyte-specific GRK2 transgenic mice did not share the same phenotype of TgGRK5 mice and did not have increased cardiac leukocyte migration and cytokine or chemokine production post-MI. Conclusions Our study shows that myocyte GRK5 has a crucial and GRK-selective role on the regulation of leucocyte infiltration into the heart, cardiac function and survival in a murine model of post-ischemic HF, supporting GRK5 inhibition as a therapeutic target for HF., Graphical Abstract

Visual Abstract, Highlights • To study the role of CTGF in post-MI cardiac repair and LV remodeling, we antagonized the function of CTGF with a mAb. • Treatment of mice with CTGF mAb during post-MI cardiac repair improved survival and resulted in better preserved LV systolic function. • Treatment with CTGF mAb during post-MI LV remodeling reduced the heart weight to body weight ratio, LV mass, cardiomyocyte hypertrophy, and fibrosis in the remote nonischemic myocardium. • CTGF mAb treatment induced c-Jun N-terminal kinase phosphorylation in ischemic hearts in vivo and in cultured human cardiac fibroblasts. • In conclusion, treatment of mice with CTGF mAb in a model of MI enhances cardiac repair and reduces adverse post-MI LV remodeling., Summary Myocardial infarction (MI)−induced cardiac fibrosis attenuates cardiac contractile function, and predisposes to arrhythmias and sudden cardiac death. Expression of connective tissue growth factor (CTGF) is elevated in affected organs in virtually every fibrotic disorder and in the diseased human myocardium. Mice were subjected to treatment with a CTGF monoclonal antibody (mAb) during infarct repair, post-MI left ventricular (LV) remodeling, or acute ischemia−reperfusion injury. CTGF mAb therapy during infarct repair improved survival and reduced LV dysfunction, and reduced post-MI LV hypertrophy and fibrosis. Mechanistically, CTGF mAb therapy induced expression of cardiac developmental and/or repair genes and attenuated expression of inflammatory and/or fibrotic genes.

Circular RNAs are generated from many protein-coding genes, but their role in cardiovascular health and disease states remains unknown. Here we report identification of circRNA transcripts that are differentially expressed in post myocardial infarction (MI) mouse hearts including circFndc3b which is significantly down-regulated in the post-MI hearts. Notably, the human circFndc3b ortholog is also significantly down-regulated in cardiac tissues of ischemic cardiomyopathy patients. Overexpression of circFndc3b in cardiac endothelial cells increases vascular endothelial growth factor-A expression and enhances their angiogenic activity and reduces cardiomyocytes and endothelial cell apoptosis. Adeno-associated virus 9 -mediated cardiac overexpression of circFndc3b in post-MI hearts reduces cardiomyocyte apoptosis, enhances neovascularization and improves left ventricular functions. Mechanistically, circFndc3b interacts with the RNA binding protein Fused in Sarcoma to regulate VEGF expression and signaling. These findings highlight a physiological role for circRNAs in cardiac repair and indicate that modulation of circFndc3b expression may represent a potential strategy to promote cardiac function and remodeling after MI.

Stem cell therapy is a potentially effective and promising treatment for ischemic heart disease. Resistin, a type of adipokine, has been found to bind to adipose-derived mesenchymal stem cells (ADSCs). However, the effects of resistin on cardiac homing by ADSCs and on ADSC-mediated cardioprotective effects have not been investigated. ADSCs were obtained from enhanced green fluorescent protein transgenic mice. C57BL/6J mice were subjected to myocardial ischemia-reperfusion (I/R) or sham operations. Six hours after the I/R operation, mice were intravenously injected with resistin-treated ADSCs (ADSC-resistin) or vehicle-treated ADSCs (ADSC-vehicle). Cardiac homing by ADSCs and cardiomyocyte apoptosis were investigated 3 days after I/R. Cardiac function, fibrosis, and angiogenesis were evaluated 4 wk after I/R. Cellular and molecular mechanisms were investigated in vitro using cultured ADSCs. Both immunostaining and flow cytometric experiments showed that resistin treatment promoted ADSC myocardial homing 3 days after intravenous injection. Echocardiographic experiments showed that ADSC-resistin, but not ADSC-vehicle, significantly improved left ventricular ejection fraction. ADSC-resistin transplantation significantly mitigated I/R-induced fibrosis and reduced atrial natriuretic peptide/brain natriuretic peptide mRNA expression. In addition, cardiomyocyte apoptosis was reduced, whereas angiogenesis was increased by ADSC-resistin treatment. At the cellular level, resistin promoted ADSC proliferation and migration but did not affect H2O2-induced apoptosis. Molecular experiments identified the ERK1/2-matrix metalloproteinase-9 pathway as a key component mediating the effects of resistin on ADSC proliferation and migration. These results demonstrate that resistin can promote homing of injected ADSCs into damaged heart tissue and stimulate functional recovery, an effect mediated through the ERK1/2 signaling pathway and matrix metalloproteinase-9. NEW & NOTEWORTHY First, intravenous injection of adipose-derived mesenchymal stem cells (ADSCs) treated with resistin significantly increased angiogenesis and reduced myocardial apoptosis and fibrosis in a murine model of ischemia-reperfusion, resulting in improved cardiac performance. Second, resistin treatment significantly increased myocardial homing of intravenously delivered ADSCs. Finally, the ERK1/2-matrix metalloproteinase 9 pathway contributed to the higher proliferative and migratory capacities of ADSCs treated with resistin.

Myocardial infarction (MI) is a leading cause of death worldwide for which there is no cure. Although cardiac cell death is a well-recognized pathological mechanism of MI, therapeutic blockade of cell death to treat MI is not straightforward. Death receptor 5 (DR5) and its ligand TRAIL [tumor necrosis factor (TNF)–related apoptosis- inducing ligand] are up-regulated in MI, but their roles in pathological remodeling are unknown. Here, we report that blocking TRAIL with a soluble DR5 immunoglobulin fusion protein diminished MI by preventing cardiac cell death and inflammation in rats, pigs, and monkeys. Mechanistically, TRAIL induced the death of cardiomyocytes and recruited and activated leukocytes, directly and indirectly causing cardiac injury. Transcriptome profiling revealed increased expression of inflammatory cytokines in infarcted heart tissue, which was markedly reduced by TRAIL blockade. Together, our findings indicate that TRAIL mediates MI directly by targeting cardiomyocytes and indirectly by affecting myeloid cells, supporting TRAIL blockade as a potential therapeutic strategy for treating MI.

Nitric oxide (NO) and S-nitrosothiol (SNO) are considered cardio- and vasoprotective substances. We now understand that one mechanism in which NO/SNOs provide cardiovascular protection is through their direct inhibition of cardiac G protein-coupled receptor (GPCR) kinase 2 (GRK2) activity via S-nitrosylation of GRK2 at cysteine 340 (C340). This maintains GPCR homeostasis, including β-adrenergic receptors, through curbing receptor GRK2-mediated desensitization. Previously, we have developed a knockin mouse (GRK2-C340S) where endogenous GRK2 is resistant to dynamic S-nitrosylation, which led to increased GRK2 desensitizing activity. This unchecked regulation of cardiac GRK2 activity resulted in significantly more myocardial damage after ischemic injury that was resistant to NO-mediated cardioprotection. Although young adult GRK2-C340S mice show no overt phenotype, we now report that as these mice age, they develop significant cardiovascular dysfunction due to the loss of SNO-mediated GRK2 regulation. This pathological phenotype is apparent as early as 12 mo of age and includes reduced cardiac function, increased cardiac perivascular fibrosis, and maladaptive cardiac hypertrophy, which are common maladies found in patients with cardiovascular disease (CVD). There are also vascular reactivity and aortic abnormalities present in these mice. Therefore, our data demonstrate that a chronic and global increase in GRK2 activity is sufficient to cause cardiovascular remodeling and dysfunction, likely due to GRK2’s desensitizing effects in several tissues. Because GRK2 levels have been reported to be elevated in elderly CVD patients, GRK2-C340 mice can give insight into the aged-molecular landscape leading to CVD. NEW & NOTEWORTHY Research on G protein-coupled receptor kinase 2 (GRK2) in the setting of cardiovascular aging is largely unknown despite its strong established functions in cardiovascular physiology and pathophysiology. This study uses a mouse model of chronic GRK2 overactivity to further investigate the consequences of long-term GRK2 on cardiac function and structure. We report for the first time that chronic GRK2 overactivity was able to cause cardiac dysfunction and remodeling independent of surgical intervention, highlighting the importance of GRK activity in aged-related heart disease.

BACKGROUND: Acute myocardial infarction (MI) and the ensuing ischemic heart disease are approaching epidemic state. Unfortunately, no definitive therapies are available and human regenerative therapies have conflicting results. Limited stem cell retention following intracoronary administration has reduced the clinical efficacy of this novel therapy. Cathelicidin related antimicrobial peptides (CRAMPs) enhance chemotactic responsiveness of BMSPCs to low SDF-1 gradients, suggesting a potential role in BMSPCs engraftment. Here, we assessed the therapeutic efficacy of CRAMPs in the context of BMSPCs recruitment and retention via intracardiac delivery of CRAMP-treated BMSPCs or CRAMP-releasing hydrogels (HG) post-AMI. METHODS: For cell transplantation experiments, mice were randomized into 3 groups: MI followed by injection of PBS, BMMNCs alone, and BMMNCs pre-incubated with CRAMP. During the in vivo HG studies, BM GFP chimera mice were randomized into 4 groups: MI followed by injection of HG alone, HG + SDF-1, HG + CRAMP, HG + SDF-1 + CRAMP. Changes in cardiac function at 5 weeks after MI were assessed using echocardiography. Angiogenesis was assessed using isolectin staining for capillary density. RESULTS: Mice treated with BMMNCs pre-incubated with CRAMP had smaller scars, enhanced cardiac recovery and less adverse remodeling. Histologically, this group had higher capillary density. Similarly, sustained CRAMP release from hydrogels enhanced the therapeutic effect of SDF-1, leading to enhanced functional recovery, smaller scar size and higher capillary density. CONCLUSION: Cathelicidins enhance BMMNC retention and recruitment after intramyocardial administration post-AMI resulting in improvements in heart physiology and recovery. Therapies employing these strategies may represent an attractive method for improving outcomes of regenerative therapies in human studies.

Visual Abstract, Highlights • Rad-GTPase is an LTCC component that functions to govern calcium current in the myocardium. • Deletion of Rad increases myocardial contractility secondary to increased trigger calcium entry. • AMI induces heart failure, including reduced calcium homeostasis, but deletion of Rad prevents AMI myocardial calcium alterations. • Rad deletion prevents post-MI scar spread by attenuating the inflammatory response. • Future studies will explore whether Rad deletion is an effective therapeutic direction for providing combined safe, stable inotropic support to the failing heart in concert with protection against inflammatory signaling., Summary The protein Rad interacts with the L-type calcium channel complex to modulate trigger Ca2+ and hence to govern contractility. Reducing Rad levels increases cardiac output. Ablation of Rad also attenuated the inflammatory response following acute myocardial infarction. Future studies to target deletion of Rad in the heart could be conducted to establish a novel treatment paradigm whereby pathologically stressed hearts would be given safe, stable positive inotropic support without arrhythmias and without pathological structural remodeling. Future investigations will also focus on establishing inhibitors of Rad and testing the efficacy of Rad deletion in cardioprotection relative to the time of onset of acute myocardial infarction.

Background: Cell therapy remains the most promising approach against ischemic heart injury. However, the poor survival of engrafted stem cells in the ischemic environment limits their therapeutic efficacy for cardiac repair after myocardial infarction. CTRP9 (C1q/tumor necrosis factor–related protein-9) is a novel prosurvival cardiokine with significantly downregulated expression after myocardial infarction. Here we tested a hypothesis that CTRP9 might be a cardiokine required for a healthy microenvironment promoting implanted stem cell survival and cardioprotection. Methods: Mice were subjected to myocardial infarction and treated with adipose-derived mesenchymal stem cells (ADSCs, intramyocardial transplantation), CTRP9, or their combination. Survival, cardiac remodeling and function, cardiomyocytes apoptosis, and ADSCs engraftment were evaluated. Whether CTRP9 directly regulates ADSCs function was determined in vitro. Discovery-drive approaches followed by cause-effect analysis were used to uncover the molecular mechanisms of CTRP9. Results: 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 P Conclusions: We provide the first evidence that CTRP9 promotes ADSCs proliferation/survival, stimulates ADSCs migration, and attenuates cardiomyocyte cell death by previously unrecognized signaling mechanisms. These include binding with N-cadherin, activation of ERK-matrix metallopeptidase 9 and ERK-nuclear factor erythroid-derived 2—like 2 signaling, and upregulation/secretion of antioxidative proteins. These results suggest that CTRP9 is a cardiokine critical in maintaining a healthy microenvironment facilitating stem cell engraftment in infarcted myocardial tissue, thereby enhancing stem cell therapeutic efficacy.

Rationale: Mesenchymal stromal cell–based therapy is promising against ischemic heart failure. However, its efficacy is limited due to low cell retention and poor paracrine function. A transmembrane protein capable of enhancing cell-cell adhesion, N-cadherin garnered attention in the field of stem cell biology only recently. Objective: The current study investigates whether and how N-cadherin may regulate mesenchymal stromal cells retention and cardioprotective capability against ischemic heart failure. Methods and Results: Adult mice–derived adipose tissue–derived mesenchymal stromal cells (ADSC) were transfected with adenovirus harboring N-cadherin, T-cadherin, or control adenovirus. CM-DiI-labeled ADSC were intramyocardially injected into the infarct border zone at 3 sites immediately after myocardial infarction (MI) or myocardial ischemia/reperfusion. ADSC retention/survival, cardiomyocyte apoptosis/proliferation, capillary density, cardiac fibrosis, and cardiac function were determined. Discovery-driven/cause-effect analysis was used to determine the molecular mechanisms. Compared with ADSC transfected with adenovirus-control, N-cadherin overexpression (but not T-cadherin) markedly increased engrafted ADSC survival/retention up to 7 days post-MI. Histological analysis revealed that ADSC transfected with adenovirus-N-cadherin significantly preserved capillary density and increased cardiomyocyte proliferation and moderately reduced cardiomyocyte apoptosis 3 days post-MI. More importantly, ADSC transfected with adenovirus-N-cadherin (but not ADSC transfected with adenovirus-T-cadherin) significantly increased left ventricular ejection fraction and reduced fibrosis in both MI and myocardial ischemia/reperfusion mice. In vitro experiments demonstrated that N-cadherin overexpression promoted ADSC-cardiomyocyte adhesion and ADSC migration, enhancing their capability to increase angiogenesis and cardiomyocyte proliferation. MMP (matrix metallopeptidases)-10/13 and HGF (hepatocyte growth factor) upregulation is responsible for N-cadherin’s effect upon ADSC migration and paracrine angiogenesis. N-cadherin overexpression promotes cardiomyocyte proliferation by HGF release. Mechanistically, N-cadherin overexpression significantly increased N-cadherin/β-catenin complex formation and active β-catenin levels in the nucleus. β-catenin knockdown abolished N-cadherin overexpression–induced MMP-10, MMP-13, and HGF expression and blocked the cellular actions and cardioprotective effects of ADSC overexpressing N-cadherin. Conclusions: We demonstrate for the first time that N-cadherin overexpression enhances mesenchymal stromal cells–protective effects against ischemic heart failure via β-catenin-mediated MMP-10/MMP-13/HGF expression and production, promoting ADSC/cardiomyocyte adhesion and ADSC retention.

Cardiac hypertrophy, including hypertension and valvular dysfunction, is a pathological feature of many cardiac diseases that ultimately leads to heart failure. Melatonin confers a protective role against pathological cardiac hypertrophy, but the underlying mechanisms remain elusive. In the present study, we hypothesized that melatonin protects against pressure overload-induced cardiac hypertrophy by attenuating Atg5-dependent autophagy and activating the Akt/mTOR pathway. Male C57BL/6 mice that received adenovirus carrying cardiac-specific Atg5 (under the cTNT promoter; Ad-cTNT-Atg5) underwent transverse aortic constriction (TAC) or sham operation and received an intraperitoneal injection of melatonin (10 mg/kg/d), vehicle or LY294002 (10 mg/kg/d) for 8 weeks. Melatonin treatment for 8 weeks markedly attenuated cardiac hypertrophy and restored impaired cardiac function, as indicated by a decreased HW/BW ratio, reduced cell cross-sectional area and fibrosis, downregulated the mRNA levels of ANP, BNP, and β-MHC and ameliorated adverse effects on the LVEF and LVFS. Melatonin treatment also inhibited apoptosis and alleviated autophagy dysfunction. Furthermore, melatonin inhibited Akt/mTOR pathway activation, while these effects were blocked by LY294002. In addition, the effect of melatonin regulation on TAC-induced autophagy dysfunction was inhibited by LY294002 or cardiac-specific Atg5 overexpression. As expected, Akt/mTOR pathway inhibition or cardiac-specific Atg5 overexpression restrained melatonin alleviation of pressure overload-induced cardiac hypertrophy. These results demonstrated that melatonin ameliorated pressure overload-induced cardiac hypertrophy by attenuating Atg5-dependent autophagy and activating the Akt/mTOR pathway.

Background Poor engraftment of intramyocardial stem cells limits their therapeutic efficiency against myocardial infarction ( MI )‐induced cardiac injury. Transglutaminase cross‐linked Gelatin (Col‐Tgel) is a tailorable collagen‐based hydrogel that is becoming an excellent biomaterial scaffold for cellular delivery in vivo. Here, we tested the hypothesis that Col‐Tgel increases retention of intramyocardially‐injected stem cells, and thereby reduces post‐ MI cardiac injury. Methods and Results Adipose‐derived mesenchymal stem cells ( ADSC s) were co‐cultured with Col‐Tgel in a 3‐dimensional system in vitro, and Col‐Tgel encapsulated ADSC s were observed using scanning electron microscopy and confocal microscopy. Vitality, proliferation, and migration of co‐cultured ADSC s were evaluated. In addition, mice were subjected to MI and were intramyocardially injected with ADSC s, Col‐Tgel, or a combination thereof. ADSC s engraftment, survival, cardiac function, and fibrosis were assessed. In vitro MTT and Cell Counting Kit‐8 assays demonstrated that ADSC s survive and proliferate up to 4 weeks in the Col‐Tgel. In addition, MTT and transwell assays showed that ADSC s migrate outside the edge of the Col‐Tgel sphere. Furthermore, when compared with ADSC s alone, Col‐Tgel‐encapsulated ADSC s significantly enhanced the long‐term retention and cardioprotective effect of ADSC s against MI ‐induced cardiac injury. Conclusions In the current study, we successfully established a 3‐dimensional co‐culture system using ADSC s and Col‐Tgel. The Col‐Tgel creates a suitable microenvironment for long‐term retention of ADSC s in an ischemic area, and thereby enhances their cardioprotective effects. Taken together, this study may provide an alternative biomaterial for stem cell‐based therapy to treat ischemic heart diseases.

Diabetes mellitus, a worldwide health threat, is considered an independent risk factor for cardiovascular diseases. The overall cardiovascular risk of diabetes is similar to the one having one myocardial infarction (MI) attack although the precise impact of diabetes on MI-induced myocardial anomalies remains elusive. Given that mortality following MI is much greater in diabetic patients compared to nondiabetic patients, this study was designed to examine the effect of melatonin on MI injury-induced myocardial dysfunction in diabetes. Adult mice were made diabetic using high-fat feeding and streptozotocin (100 mg/kg body weight) prior to MI and were treated with melatonin (50 mg/kg/d, p.o.) for 4 weeks prior to assessment of cardiac geometry and function. The MI procedure in diabetes displayed overt changes in cardiac geometry (chamber dilation and interstitial fibrosis) and functional anomalies (reduced fractional shortening and cardiomyocyte contractile capacity) in association with elevated c-Jun N-terminal kinase (JNK) phosphorylation and p53 level. Melatonin treatment markedly attenuated cardiac dysfunction and myocardial fibrosis in post-MI diabetic mice. Furthermore, melatonin decreased JNK phosphorylation, reduced p53 levels, and suppressed apoptosis in hearts from the post-MI diabetic group. In vitro findings revealed that melatonin effectively counteracted high-glucose/high fat-hypoxia-induced cardiomyocyte apoptosis and contractile dysfunction through a JNK-mediated mechanism, the effects of which were impaired by the JNK activator anisomycin. In summary, our study suggests that melatonin protects against myocardial injury in post-MI mice with diabetes, which offers a new therapeutic strategy for the management of MI-induced cardiac injury in diabetes.

Objective: Local and systemic metabolic disorders during heart failure (HF) affecting the disease progression and prognosis. The long-range organ-to-organ communications mediates the coordination of organs homeostasis and adaptation. However, it remains unknown whether and how the failing heart contributes to the adipose dysfunction. Method and Results: 6-8 week-old adult wild type (WT) male mice were subjected to myocardial infraction (MI) for 8 weeks. The cardiac function was evaluated by echocardiography. The plasma lipid profiles were evaluated in Sham and HF mice via Alere LDX analyzer. Interestingly, white adipose tissue weight was significantly decreased, TC/HDL ratio and LDL levels were increased in HF mice compared to non-MI sham mice. To determine the possible pathological mediator, the sEV of murine serum, heart tissue perfusate and cardiomyocyte culture medium from HF mice were isolated and incubated with adipocytes (3T3L1). The adipogenesis was significantly inhibited when sEV isolated from three different resources were administrated to 3T3L1 adipocytes. Pathway specific discover-driven approach revealed that Pparg, Cebpa and Cebpd expression were significantly inhibited by sEV derived from HF cardiomyocytes. To determine the specific mediators in sEV, Venn diagraph assay were performed with two databases (the cardiomyocyte-enriched miRNAs and adipocyte development-related miRNAs). The cluster of miR-23-27-24 and the miR-30 family were markedly elevated in serum-derived, heart tissue-derived and cardiomyocyte-derived sEV from MI mice compared to Sham mice. These changes were validated by RT-qPCR assay. Treatment with miR-23-27-24 significantly inhibited adipogenesis. Conclusion: Taken together, microRNAs miR-23-27-24 and the miR-30 in cardiac-derived sEV are the key messengers facilitating adipocyte dysfunction after MI. Adiogenesis suppression is regulated by failing myocardium-derived sEV, which results in progressive matabolic disorders in the adipose tissue.

Circular RNAs are generated from many protein-coding genes, but their role in cardiovascular health and disease states remains unknown. Here we report identification of circRNA transcripts that are differentially expressed in post myocardial infarction (MI) mouse hearts including circFndc3b which is significantly down-regulated in the post-MI hearts. Notably, the human circFndc3b ortholog is also significantly down-regulated in cardiac tissues of ischemic cardiomyopathy patients. Overexpression of circFndc3b in cardiac endothelial cells increases vascular endothelial growth factor-A expression and enhances their angiogenic activity and reduces cardiomyocytes and endothelial cell apoptosis. Adeno-associated virus 9 -mediated cardiac overexpression of circFndc3b in post-MI hearts reduces cardiomyocyte apoptosis, enhances neovascularization and improves left ventricular functions. Mechanistically, circFndc3b interacts with the RNA binding protein Fused in Sarcoma to regulate VEGF expression and signaling. These findings highlight a physiological role for circRNAs in cardiac repair and indicate that modulation of circFndc3b expression may represent a potential strategy to promote cardiac function and remodeling after MI., Circular RNAs (circRNAs) are non-coding RNAs generated from pre-mRNAs of coding genes by the splicing machinery whose function in the heart is poorly understood. Here the authors show that AAV-mediated delivery of the circRNA circFndc3b prevents cardiomyocyte apoptosis, enhances angiogenesis, and attenuates LV dysfunction post-MI in mice by regulating FUS-VEGF-A signalling.

By incompletely understood mechanisms, MI patients sustain systemic metabolic disorder. Adipocytes are an important cellular type regulating energy homeostasis. The impact of MI upon adipocyte function remains unknown. Exosomes (Exo) are critical vehicles mediating organ-organ communication. However, whether and how Exo may mediate post-MI cardiomyocyte/adipocyte communication have not been previously investigated. Adult male mice were subjected to MI/R. Serum Exo were isolated 3 hours after R and incubated with 3T3L cells for 24 hours. Compared to control, MI/R Exo significantly altered the expression of 17 genes known to be important in adipocyte function. GO analysis revealed that genes associated with endoplasmic reticulum (ER) function and adipocyte endocrine function are the primary two pathways altered by MI/R Exo. Venn analysis identified 11 mi-RNAs as cardiac-enriched, adipocyte-poor, and ER function-related miRNAs. RT-qPCR confirmed the miR-23a/27a/24-2 family members are the most markedly increased mi-RNAs in MI/R Exo. Incubation of 3T3L cells with mi-R27a mimic significantly downregulated EDEM3, DsBA-L, and PPARγ, and upregulated PERK and CHOP. Conversely, mi-R27a inhibitor significantly decreased the impact of MI/R Exo upon ER function genes. Additional studies demonstrated EDEM3 and PPARγ (two critical molecules maintaining ER function and adipocyte endocrine function) to be direct targets of mi-R27a. One of the most significant endocrine molecules of adipocyte origin, adiponectin is regulated by PPARγ at the transcriptional level and by DsBA-L at the post-translational level. We next determined whether MI/R Exo may affect adiponectin expression/assembly. Incubation of 3T3L cells with MI/R Exo significantly inhibited total and high molecular weight adiponectin expression, an effect blocked by miR27a mimic. Finally, in vivo administration of GW4869 (Exo biogenesis inhibitor) or miR27a inhibitor attenuated adipocyte ER dysfunction and restored plasma adiponectin level in MI/R animals. We demonstrate for the first time that MI/R causes significant adipocyte ER and endocrine dysfunction by Exo mediated cardiomyocyte/adipocyte communication via miR-23a/27a/24-2.

Mesenchymal stromal cells (MSCs) transplantation offers an attractive alternative in myocardial infarctive therapy. However, poor cell engraftment and survival limit their restorative capacity. C1q/tumor necrosis factor-related protein-3 (CTRP3) inhibits reverse remodeling after myocardial infarction (MI) and was found to be secreted by MSCs in our preliminary experiments. We examined whether the overexpression of CTRP3 improved the survival of transplanted MSCs and augmented their efficacy on MI and whether silencing CTRP3 attenuated these effects. For gain-of-function analysis, MSCs overexpressing CTRP3 (LvC3-MSCs), control virus-transfected MSCs (LvNull-MSCs), MSCs alone, or phosphate-buffered saline (PBS) were injected into the peripheral areas of the infarction immediately after coronary artery ligation. For loss-of-function analysis, mice subjected to MI were randomized into groups and administered CTRP3-knockdown MSCs (LvshC3-MSCs), Lvshctrl-MSCs, MSCs, or PBS. Survival rates, cardiac function, and myocardial remodeling in mice were evaluated after 4 weeks. Injection of MSCs or LvNull-MSCs improved the left ventricular ejection fraction, inhibited cardiac fibrosis, and regulated cellular profiles of the infarction border zone 4 weeks after MI compared with those in the PBS group. Furthermore, overexpression of hCTRP3 promoted the efficacy of MSCs in the treatment of MI. However, knocking down CTRP3 impaired that. Coculture experiments confirmed that hCTRP3-enriched conditioned medium (CM) promoted MSCs migration and protected against H2O2-induced cell damage. Conversely, CM from C3−/− MSCs (CTRP3 knock out) significantly reduced the migration and antioxidative effects of MSCs. CTRP3 protein alone promoted MSCs proliferation and migration by upregulating matrix metalloproteinase 9 (MMP9) and protecting against oxidation by increasing superoxide dismutase 2 (SOD2) and metallothionein 1/2 (MT1/2) expression; and these effects were blocked by pretreatment with the extracellular signal-regulated kinase (ERK1/2) inhibitor U0126. Overexpression of CTRP3 significantly improved the MSCs-based efficacy on MI by increasing cell survival and retention via a mechanism involving ERK1/2-MMP9 and ERK1/2-SOD2/MT1/2 signaling.

Following injury, leukocytes are released from hematopoietic organs and migrate to the site of damage to regulate tissue inflammation and repair; however, leukocytes lacking beta(2)-adrenergic receptor (beta(2)-AR) expression have marked impairments in these processes. Beta blockade is a common strategy for the treatment of many cardiovascular etiologies; therefore, the objective of our study was to assess the impact of prior beta blacker treatment on baseline leukocyte parameters and their responsiveness to acute injury. In a temporal and beta-adrenergic receptor isoform-dependent manner, chronic beta blocker infusion increased splenic vascular cell adhesion molecule 1 expression and leukocyte accumulation (monocytes/macrophages, mast cells, and neutrophils) and decreased chemokine receptor 2 (CCR2) expression and migration of bone marrow and peripheral blood leukocytes (PBLs) to, as well as infiltration into, the heart following acute cardiac injury. Further, CCR2 expression and migratory responsiveness were significantly reduced in the PBLs of patients receiving beta blocker therapy compared with beta blocker-naive patients. These results highlight the ability of chronic beta blocker treatment to alter baseline leukocyte characteristics that decrease leukocytes' responsiveness to acute injury and suggest that prior beta blockade may act to reduce the severity of innate immune responses.

The present study was undertaken to test the hypothesis that gene delivery of BCL2-Associated Athanogene 3 (BAG3) to the heart of mice with left ventricular dysfunction secondary to a myocardial infarction could enhance cardiac performance.BAG3 is a 575 amino acid protein that has pleotropic functions in the cell including pro-autophagy and anti-apoptosis. Mutations in BAG3 have been associated with both skeletal muscle dysfunction and familial dilated cardiomyopathy and BAG3 levels are diminished in non-familial heart failure.Eight-week-old C57/BL6 mice underwent ligation of the left coronary artery (MI) or sham surgery (Sham). Eight weeks later, mice in both groups were randomly assigned to receive either a retro-orbital injection of rAAV9-BAG3 (MI-BAG3 or Sham-BAG3) or rAAV9-GFP (MI-GFP or Sham GFP). Mice were sacrificed at 3 weeks post-injection and myocytes were isolated from the left ventricle.MI-BAG3 mice demonstrated a significantly (p0.0001) higher left ventricular ejection fraction (LVEF) 9 days after rAAV9-BAG3 injection with further improvement in LVEF, fractional shortening and stroke volume at 3 weeks post-injection without changes in LV mass or LV volume. Injection of rAAV9-BAG3 had no effect on LVEF in Sham mice. The salutary benefits of rAAV9-BAG3 were also observed in myocytes isolated from MI hearts including improved cell shortening (p0.05), increased systolic [CaThe results suggest that BAG3 gene therapy may provide a novel therapeutic option for the treatment of heart failure.

Rationale: G protein–coupled receptor kinase 2 (GRK2) is an important molecule upregulated after myocardial injury and during heart failure. Myocyte-specific GRK2 loss before and after myocardial ischemic injury improves cardiac function and remodeling. The cardiac fibroblast plays an important role in the repair and remodeling events after cardiac ischemia; the importance of GRK2 in these events has not been investigated. Objective: The aim of this study is to elucidate the in vivo implications of deleting GRK2 in the cardiac fibroblast after ischemia/reperfusion injury. Methods and Results: We demonstrate, using Tamoxifen inducible, fibroblast-specific GRK2 knockout mice, that GRK2 loss confers a protective advantage over control mice after myocardial ischemia/reperfusion injury. Fibroblast GRK2 knockout mice presented with decreased infarct size and preserved cardiac function 24 hours post ischemia/reperfusion as demonstrated by increased ejection fraction (59.1±1.8% versus 48.7±1.2% in controls; P Conclusions: These novel data showing the benefits of inhibiting GRK2 in the cardiac fibroblast adds to previously published data showing the advantage of GRK2 ablation and reinforces the therapeutic potential of GRK2 inhibition in the heart after myocardial ischemia.

Vasopressin type 1A receptor (V1AR) expression is elevated in chronic human heart failure (HF) and contributes to cardiac dysfunction in animal models, in part via reduced β-adrenergic receptor (βAR) responsiveness. Although cardiac V1AR overexpression and V1AR stimulation are each sufficient to decrease βAR activity, it is unknown whether V1AR inhibition conversely augments βAR responsiveness. Further, although V1AR has been shown to contribute to chronic progression of HF, its impact on cardiac function following acute ischaemic injury has not been reported. Using V1AR knockout (V1AR KO) mice we assessed the impact of V1AR deletion on cardiac contractility at baseline and following ischaemic injury, βAR sensitivity and cardiomyocyte responsiveness to βAR stimulation. Strikingly, baseline cardiac contractility was enhanced in V1AR KO mice and they experienced a greater loss in contractile function than control mice following acute ischaemic injury, although the absolute levels of cardiac dysfunction and survival rates did not differ. Enhanced cardiac contractility in V1AR KO mice was associated with augmented β-blocker sensitivity, suggesting increased basal βAR activity, and indeed levels of left ventricular cAMP, as well as phospholamban (PLB) and cardiac troponin I (cTnI) phosphorylation were elevated compared with control mice. At the cellular level, myocytes isolated from V1AR KO mice demonstrated increased responsiveness to βAR stimulation consistent with the finding that acute pharmacological V1AR inhibition enhanced βAR-mediated contractility in control myocytes. Therefore, although V1AR deletion does not protect the heart from the rapid development of cardiac dysfunction following acute ischaemic injury, its effects on βAR activity suggest that acute V1AR inhibition could be utilized to promote myocyte contractile performance.

Background: Immune cell–mediated inflammation is an essential process for mounting a repair response after myocardial infarction (MI). The sympathetic nervous system is known to regulate immune system function through β-adrenergic receptors (βARs); however, their role in regulating immune cell responses to acute cardiac injury is unknown. Methods: Wild-type (WT) mice were irradiated followed by isoform-specific βAR knockout (βARKO) or WT bone-marrow transplantation (BMT) and after full reconstitution underwent MI surgery. Survival was monitored over time, and alterations in immune cell infiltration after MI were examined through immunohistochemistry. Alterations in splenic function were identified through the investigation of altered adhesion receptor expression. Results: β 2 ARKO BMT mice displayed 100% mortality resulting from cardiac rupture within 12 days after MI compared with ≈20% mortality in WT BMT mice. β 2 ARKO BMT mice displayed severely reduced post-MI cardiac infiltration of leukocytes with reciprocally enhanced splenic retention of the same immune cell populations. Splenic retention of the leukocytes was associated with an increase in vascular cell adhesion molecule-1 expression, which itself was regulated via β-arrestin–dependent β 2 AR signaling. Furthermore, vascular cell adhesion molecule-1 expression in both mouse and human macrophages was sensitive to β 2 AR activity, and spleens from human tissue donors treated with β-blocker showed enhanced vascular cell adhesion molecule-1 expression. The impairments in splenic retention and cardiac infiltration of leukocytes after MI were restored to WT levels via lentiviral-mediated re-expression of β 2 AR in β 2 ARKO bone marrow before transplantation, which also resulted in post-MI survival rates comparable to those in WT BMT mice. Conclusions: Immune cell–expressed β 2 AR plays an essential role in regulating the early inflammatory repair response to acute myocardial injury by facilitating cardiac leukocyte infiltration.

Activin A and myostatin, members of the transforming growth factor (TGF)-β superfamily of secreted factors, are potent negative regulators of muscle growth, but their contribution to myocardial ischemia-reperfusion (IR) injury is not known. The aim of this study was to investigate if activin 2B (ACVR2B) receptor ligands contribute to myocardial IR injury. Mice were treated with soluble ACVR2B decoy receptor (ACVR2B-Fc) and subjected to myocardial ischemia followed by reperfusion for 6 or 24 h. Systemic blockade of ACVR2B ligands by ACVR2B-Fc was protective against cardiac IR injury, as evidenced by reduced infarcted area, apoptosis, and autophagy and better preserved LV systolic function following IR. ACVR2B-Fc modified cardiac metabolism, LV mitochondrial respiration, as well as cardiac phenotype toward physiological hypertrophy. Similar to its protective role in IR injury in vivo, ACVR2B-Fc antagonized SMAD2 signaling and cell death in cardiomyocytes that were subjected to hypoxic stress. ACVR2B ligand myostatin was found to exacerbate hypoxic stress. In addition to acute cardioprotection in ischemia, ACVR2B-Fc provided beneficial effects on cardiac function in prolonged cardiac stress in cardiotoxicity model. By blocking myostatin, ACVR2B-Fc potentially reduces cardiomyocyte death and modifies cardiomyocyte metabolism for hypoxic conditions to protect the heart from IR injury., Graphical Abstract, Magga et al. report that systemic blockade of activin receptor type 2 (ACVR2B) ligands protects from cardiac ischemia-reperfusion injury reducing infarct size, apoptosis, and autophagy. Mechanistically, systemic blockade of ACVR2B ligands reduced SMAD2 activation and modified cardiomyocyte metabolism for hypoxic conditions.