Nicotinamide mononucleotide as a therapeutic agent to alleviate multi-organ failure in sepsis.

BACKGROUND: Sepsis-caused multi-organ failure remains the major cause of morbidity and mortality in intensive care units with limited therapeutics. Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD+), has been recently reported to be protective in sepsis; however, its therapeutic effects remain to be determined. This study sought to investigate the therapeutic effects of NMN in septic organ failure and its underlying mechanisms. METHODS: Sepsis was induced by feces-injection-in-peritoneum in mice. NMN was given after an hour of sepsis onset. Cultured neutrophils, macrophages and endothelial cells were incubated with various agents. RESULTS: We demonstrate that administration of NMN elevated NAD+ levels and reduced serum lactate levels, oxidative stress, inflammation, and caspase-3 activity in multiple organs of septic mice, which correlated with the attenuation of heart dysfunction, pulmonary microvascular permeability, liver injury, and kidney dysfunction, leading to lower mortality. The therapeutic effects of NMN were associated with lower bacterial burden in blood, and less ROS production in septic mice. NMN improved bacterial phagocytosis and bactericidal activity of macrophages and neutrophils while reducing the lipopolysaccharides-induced inflammatory response of macrophages. In cultured endothelial cells, NMN mitigated mitochondrial dysfunction, inflammation, apoptosis, and barrier dysfunction induced by septic conditions, all of which were offset by SIRT3 inhibition. CONCLUSION: NAD+ repletion with NMN prevents mitochondrial dysfunction and restrains bacterial dissemination while limiting inflammatory damage through SIRT3 signaling in sepsis. Thus, NMN may represent a therapeutic option for sepsis.

MLKL-mediated necroptosis is a target for cardiac protection in mouse models of type-1 diabetes.

BACKGROUND: Cardiomyocyte death contributes to cardiac pathology of diabetes. Studies have shown that the RIPK3/MLKL necroptosis signaling is activated in diabetic hearts. Deletion of RIPK3 was reported to attenuate myocardial injury and heart dysfunction in streptozocin (STZ)-induced diabetic mice, suggesting a potential role of necroptosis in diabetic cardiomyopathy. This study characterized cardiomyocyte necroptosis in diabetic hearts and investigated whether MLKL-mediated necroptosis is a target for cardiac protection in diabetes. METHODS: Type 1 diabetes was induced in RIPK3 knockout, MLKL knockout and wild-type mice. Akita Type-1 diabetic mice were injected with shRNA for MLKL. Myocardial function was assessed by echocardiography. Immuno-histological analyses determined cardiomyocyte death and fibrosis in the heart. Cultured adult mouse cardiomyocytes were incubated with high glucose in the presence of various drugs. Cell death and phosphorylation of RIPK3 and MLKL were analysed. RESULTS: We showed that the levels of phosphorylated RIPK3 and MLKL were higher in high glucose-stimulated cardiomyocytes and hearts of STZ-induced type-1 diabetic mice, akita mice and type-1 diabetic monkeys when compared to non-diabetic controls. Inhibition of RIPK3 by its pharmacological inhibitor or gene deletion, or MLKL deletion prevented high glucose-induced MLKL phosphorylation and attenuated necroptosis in cardiomyocytes. In STZ-induced type-1 diabetic mice, cardiomyocyte necroptosis was present along with elevated cardiac troponin I in serum and MLKL oligomerization, and co-localized with phosphorylated MLKL. Deletion of RIPK3 or MLKL prevented MLKL phosphorylation and cardiac necroptosis, attenuated serum cardiac troponin I levels, reduced myocardial collagen deposition and improved myocardial function in STZ-injected mice. Additionally, shRNA-mediated down-regulation of MLKL reduced cardiomyocyte necroptosis in akita mice. Interestingly, incubation with anti-diabetic drugs (empagliflozin and metformin) prevented phosphorylation of RIPK3 and MLKL, and reduced cell death in high glucose-induced cardiomyocytes. CONCLUSIONS: We have provided evidence that cardiomyocyte necroptosis is present in diabetic hearts and that MLKL-mediated cardiomyocyte necroptosis contributes to diabetic cardiomyopathy. These findings highlight MLKL-mediated necroptosis as a target for cardiac protection in diabetes.

Identification of a Novel Antisepsis Pathway: Sectm1a Enhances Macrophage Phagocytosis of Bacteria through Activating GITR.

The inability to effectively control invading bacteria or other pathogens is a major cause of multiple organ dysfunction and death in sepsis. As the first-line defense of the immune system, macrophages play a crucial role in the removal of pathogens during sepsis. In this study, we define secreted and transmembrane 1A (Sectm1a) as a novel ligand of glucocorticoid-induced TNFR (GITR) that greatly boosts macrophage phagocytosis and bactericidal capacity. Using a global Sectm1a knockout (KO) mouse model, we observed that Sectm1a deficiency significantly suppressed phagocytosis and bactericidal activity in both recruited macrophages and tissue-resident macrophages, which consequently aggravated bacterial burden in the blood and multiple organs and further increased systemic inflammation, leading to multiple organ injury and increased mortality during polymicrobial sepsis. By contrast, treatment of septic mice with recombinant Sectm1a protein (rSectm1a) not only promoted macrophage phagocytosis and bactericidal activity but also significantly improved survival outcome. Mechanistically, we identified that Sectm1a could bind to GITR in the surface of macrophages and thereby activate its downstream PI3K-Akt pathway. Accordingly, rSectm1a-mediated phagocytosis and bacterial killing were abolished in macrophages by either KO of GITR or pharmacological inhibition of the PI3K-Akt pathway. In addition, rSectm1a-induced therapeutic effects on sepsis injury were negated in GITR KO mice. Taken together, these results uncover that Sectm1a may represent a novel target for drug development to control bacterial dissemination during sepsis or other infectious diseases.

Sustained over-expression of calpain-2 induces age-dependent dilated cardiomyopathy in mice through aberrant autophagy.

Calpains have been implicated in heart diseases. While calpain-1 has been detrimental to the heart, the role of calpain-2 in cardiac pathology remains controversial. In this study we investigated whether sustained over-expression of calpain-2 had any adverse effects on the heart and the underlying mechanisms. Double transgenic mice (Tg-Capn2/tTA) were generated, which express human CAPN2 restricted to cardiomyocytes. The mice were subjected to echocardiography at age 3, 6, 8 and 12 months, and their heart tissues and sera were collected for analyses. We showed that transgenic mice over-expressing calpain-2 restricted to cardiomyocytes had normal heart function with no evidence of cardiac pathological remodeling at age 3 months. However, they exhibited features of dilated cardiomyopathy including increased heart size, enlarged heart chambers and heart dysfunction from age 8 months; histological analysis revealed loss of cardiomyocytes replaced by myocardial fibrosis and cardiomyocyte hypertrophy in transgenic mice from age 8 months. These cardiac alterations closely correlated with aberrant autophagy evidenced by significantly increased LC3BII and p62 protein levels and accumulation of autophagosomes in the hearts of transgenic mice. Notably, injection of 3-methyladenine, a well-established inhibitor of autophagy (30 mg/kg, i.p. once every 3 days starting from age 6 months for 2 months) prevented aberrant autophagy, attenuated myocardial injury and improved heart function in the transgenic mice. In cultured cardiomyocytes, over-expression of calpain-2 blocked autophagic flux by impairing lysosomal function. Furthermore, over-expression of calpain-2 resulted in lower levels of junctophilin-2 protein in the heart of transgenic mice and in cultured cardiomyocytes, which was attenuated by 3-methyladenine. In addition, blockade of autophagic flux by bafilomycin A (100 nM) induced a reduction of junctophilin-2 protein in cardiomyocytes. In summary, transgenic over-expression of calpain-2 induces age-dependent dilated cardiomyopathy in mice, which may be mediated through aberrant autophagy and a reduction of junctophilin-2. Thus, a sustained increase in calpain-2 may be detrimental to the heart.

Sectm1a Facilitates Protection against Inflammation-Induced Organ Damage through Promoting TRM Self-Renewal.

Tissue-resident macrophages (TRMs) are sentinel cells for maintaining tissue homeostasis and organ function. In this study, we discovered that lipopolysaccharide (LPS) administration dramatically reduced TRM populations and suppressed their self-renewal capacities in multiple organs. Using loss- and gain-of-function approaches, we define Sectm1a as a novel regulator of TRM self-renewal. Specifically, at the earlier stage of endotoxemia, Sectm1a deficiency exaggerated acute inflammation-induced reduction of TRM numbers in multiple organs by suppressing their proliferation, which was associated with more infiltrations of inflammatory monocytes/neutrophils and more serious organ damage. By contrast, administration of recombinant Sectm1a enhanced TRM populations and improved animal survival upon endotoxin challenge. Mechanistically, we identified that Sectm1a-induced upregulation in the self-renewal capacity of TRM is dependent on GITR-activated T helper cell expansion and cytokine production. Meanwhile, we found that TRMs may play an important role in protecting local vascular integrity during endotoxemia. Our study demonstrates that Sectm1a contributes to stabling TRM populations through maintaining their self-renewal capacities, which benefits the host immune response to acute inflammation. Therefore, Sectm1a may serve as a new therapeutic agent for the treatment of inflammatory diseases.

Calpain-2 specifically cleaves Junctophilin-2 at the same site as Calpain-1 but with less efficacy.

Calpain proteolysis contributes to the pathogenesis of heart failure but the calpain isoforms responsible and their substrate specificities have not been rigorously defined. One substrate, Junctophilin-2 (JP2), is essential for maintaining junctional cardiac dyads and excitation-contraction coupling. We previously demonstrated that mouse JP2 is cleaved by calpain-1 (CAPN1) between Arginine 565 (R565) and Threonine 566 (T566). Recently, calpain-2 (CAPN2) was reported to cleave JP2 at a novel site between Glycine 482 (G482) and Threonine 483 (T483). We aimed to directly compare the contributions of each calpain isoform, their Ca2+ sensitivity, and their cleavage site selection for JP2. We find CAPN1, CAPN2 and their requisite CAPNS1 regulatory subunit are induced by pressure overload stress that is concurrent with JP2 cleavage. Using in vitro calpain cleavage assays, we demonstrate that CAPN1 and CAPN2 cleave JP2 into similar 75 kD N-terminal (JP2NT) and 25 kD C-terminal fragments (JP2CT) with CAPNS1 co-expression enhancing proteolysis. Deletion mutagenesis shows both CAPN1 and CAPN2 require R565/T566 but not G482/T483. When heterologously expressed, the JP2CT peptide corresponding to R565/T566 cleavage approximates the 25 kD species found during cardiac stress while the C-terminal peptide from potential cleavage at G482/T483 produces a 35 kD product. Similar results were obtained for human JP2. Finally, we show that CAPN1 has higher Ca2+ sensitivity and cleavage efficacy than CAPN2 on JP2 and other cardiac substrates including cTnT, cTnI and ß2-spectrin. We conclude that CAPN2 cleaves JP2 at the same functionally conserved R565/T566 site as CAPN1 but with less efficacy and suggest heart failure may be targeted through specific inhibition of CAPN1.

Gamma-Aminobutyrate Transaminase Protects against Lipid Overload-Triggered Cardiac Injury in Mice.

Lipid overload contributes to cardiac complications of diabetes and obesity. However, the underlying mechanisms remain obscure. This study investigates the role of gamma-aminobutyrate transaminase (ABAT), the key enzyme involved in the catabolism of γ-aminobutyric acid (GABA), in lipid overload-induced cardiac injury. Microarray revealed a down-regulation of ABAT mRNA expression in high fat diet (HFD)-fed mouse hearts, which correlated with a reduction in ABAT protein level and its GABA catabolic activity. Transgenic mice with cardiomyocyte-specific ABAT over-expression (Tg-ABAT/tTA) were generated to determine the role of ABAT in lipid overload-induced cardiac injury. Feeding with a HFD to control mice for 4 months reduced ATP production and the mitochondrial DNA copy number, and induced myocardial oxidative stress, hypertrophy, fibrosis and dysfunction. Such pathological effects of HFD were mitigated by ABAT over-expression in Tg-ABAT/tTA mice. In cultured cardiomyocytes, palmitate increased mitochondrial ROS production, depleted ATP production and promoted apoptosis, all of which were attenuated by ABAT over-expression. With the inhibition of ABAT's GABA catabolic activity, the protective effects of ABAT remained unchanged in palmitate-induced cardiomyocytes. Thus, ABAT protects the mitochondrial function in defending the heart against lipid overload-induced injury through mechanisms independent of its GABA catabolic activity, and may represent a new therapeutic target for lipid overload-induced cardiac injury.

Calpain activation mediates microgravity-induced myocardial abnormalities in mice via p38 and ERK1/2 MAPK pathways.

The human cardiovascular system has adapted to function optimally in Earth's 1G gravity, and microgravity conditions cause myocardial abnormalities, including atrophy and dysfunction. However, the underlying mechanisms linking microgravity and cardiac anomalies are incompletely understood. In this study, we investigated whether and how calpain activation promotes myocardial abnormalities under simulated microgravity conditions. Simulated microgravity was induced by tail suspension in mice with cardiomyocyte-specific deletion of Capns1, which disrupts activity and stability of calpain-1 and calpain-2, and their WT littermates. Tail suspension time-dependently reduced cardiomyocyte size, heart weight, and myocardial function in WT mice, and these changes were accompanied by calpain activation, NADPH oxidase activation, and oxidative stress in heart tissues. The effects of tail suspension were attenuated by deletion of Capns1 Notably, the protective effects of Capns1 deletion were associated with the prevention of phosphorylation of Ser-345 on p47 phox and attenuation of ERK1/2 and p38 activation in hearts of tail-suspended mice. Using a rotary cell culture system, we simulated microgravity in cultured neonatal mouse cardiomyocytes and observed decreased total protein/DNA ratio and induced calpain activation, phosphorylation of Ser-345 on p47 phox , and activation of ERK1/2 and p38, all of which were prevented by calpain inhibitor-III. Furthermore, inhibition of ERK1/2 or p38 attenuated phosphorylation of Ser-345 on p47 phox in cardiomyocytes under simulated microgravity. This study demonstrates for the first time that calpain promotes NADPH oxidase activation and myocardial abnormalities under microgravity by facilitating p47 phox phosphorylation via ERK1/2 and p38 pathways. Thus, calpain inhibition may be an effective therapeutic approach to reduce microgravity-induced myocardial abnormalities.

Targeted inhibition of calpain in mitochondria alleviates oxidative stress-induced myocardial injury.

The protein levels and activities of calpain-1 and calpain-2 are increased in cardiac mitochondria under pathological conditions including ischemia, diabetes, and sepsis, and transgenic overexpression of mitochondrial-targeted calpain-1 induces dilated heart failure, which underscores an important role of increased calpain in mitochondria in mediating myocardial injury. However, it remains to be determined whether selective inhibition of calpain in mitochondria protects the heart under pathological conditions. In this study, we generated transgenic mice overexpressing mitochondrial-targeted calpastatin in cardiomyocytes. Their hearts were isolated and subjected to global ischemia/reperfusion. Hyperglycemia was induced in the transgenic mice by injections of STZ. We showed that transgenic calpastatin was expressed exclusively in mitochondria isolated from their hearts but not from other organs including skeletal muscle and lung tissues. Transgenic overexpression of mitochondrial-targeted calpastatin significantly attenuated mitochondrial oxidative stress and cell death induced by global ischemia/reperfusion in isolated hearts, and ameliorated mitochondrial oxidative stress, cell death, myocardial remodeling and dysfunction in STZ-treated transgenic mice. The protective effects of mitochondrial-targeted calpastatin were correlated with increased ATP5A1 protein expression and ATP synthase activity in isolated hearts subjected to global ischemia/reperfusion and hearts of STZ-treated transgenic mice. In cultured rat myoblast H9c2 cells, overexpression of mitochondrial-targeted calpastatin maintained the protein levels of ATP5A1 and ATP synthase activity, prevented mitochondrial ROS production and decreased cell death following hypoxia/reoxygenation, whereas upregulation of ATP5A1 or scavenging of mitochondrial ROS by mito-TEMPO abrogated mitochondrial ROS production and decreased cell death. These results confirm the role of calpain in myocardial injury, suggesting that selective inhibition of calpain in myocardial mitochondria by mitochondrial-targeted calpastatin is an effective strategy for alleviating myocardial injury and dysfunction in cardiac pathologies.

MicroRNA-223 is essential for maintaining functional ß-cell mass during diabetes through inhibiting both FOXO1 and SOX6 pathways.

The initiation and development of diabetes are mainly ascribed to the loss of functional ß-cells. Therapies designed to regenerate ß-cells provide great potential for controlling glucose levels and thereby preventing the devastating complications associated with diabetes. This requires detailed knowledge of the molecular events and underlying mechanisms in this disorder. Here, we report that expression of microRNA-223 (miR-223) is up-regulated in islets from diabetic mice and humans, as well as in murine Min6 ß-cells exposed to tumor necrosis factor α (TNFα) or high glucose. Interestingly, miR-223 knockout (KO) mice exhibit impaired glucose tolerance and insulin resistance. Further analysis reveals that miR-223 deficiency dramatically suppresses ß-cell proliferation and insulin secretion. Mechanistically, using luciferase reporter gene assays, histological analysis, and immunoblotting, we demonstrate that miR-223 inhibits both forkhead box O1 (FOXO1) and SRY-box 6 (SOX6) signaling, a unique bipartite mechanism that modulates expression of several ß-cell markers (pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), and urocortin 3 (UCN3)) and cell cycle-related genes (cyclin D1, cyclin E1, and cyclin-dependent kinase inhibitor P27 (P27)). Importantly, miR-223 overexpression in ß-cells could promote ß-cell proliferation and improve ß-cell function. Taken together, our results suggest that miR-223 is a critical factor for maintaining functional ß-cell mass and adaptation during metabolic stress.

Tsg101 positively regulates physiologic-like cardiac hypertrophy through FIP3-mediated endosomal recycling of IGF-1R.

Development of physiologic cardiac hypertrophy has primarily been ascribed to the IGF-1 and its receptor, IGF-1 receptor (IGF-1R), and subsequent activation of the protein kinase B (Akt) pathway. However, regulation of endosome-mediated recycling and degradation of IGF-1R during physiologic hypertrophy has not been investigated. In a physiologic hypertrophy model of treadmill-exercised mice, we observed that levels of tumor susceptibility gene 101 (Tsg101), a key member of the endosomal sorting complex required for transport, were dramatically elevated in the heart compared with sedentary controls. To determine the role of Tsg101 on physiologic hypertrophy, we generated a transgenic (TG) mouse model with cardiac-specific overexpression of Tsg101. These TG mice exhibited a physiologic-like cardiac hypertrophy phenotype at 8 wk evidenced by: 1) the absence of cardiac fibrosis, 2) significant improvement of cardiac function, and 3) increased total and plasma membrane levels of IGF-1R and increased phosphorylation of Akt. Mechanistically, we identified that Tsg101 interacted with family-interacting protein 3 (FIP3) and IGF-1R, thereby stabilizing FIP3 and enhancing recycling of IGF-1R. In vitro, adenovirus-mediated overexpression of Tsg101 in neonatal rat cardiomyocytes resulted in cell hypertrophy, which was blocked by addition of monensin, an inhibitor of endosome-mediated recycling, and by small interfering RNA-mediated knockdown (KD) of FIP3. Furthermore, cardiac-specific KD of Tsg101 showed a significant reduction in levels of endosomal recycling compartment members (Rab11a and FIP3), IGF-1R, and Akt phosphorylation. Most interestingly, Tsg101-KD mice failed to develop cardiac hypertrophy after intense treadmill training. Taken together, our data identify Tsg101 as a novel positive regulator of physiologic cardiac hypertrophy through facilitating the FIP3-mediated endosomal recycling of IGF-1R.-Essandoh, K., Deng, S., Wang, X., Jiang, M., Mu, X., Peng, J., Li, Y., Peng, T., Wagner, K.-U., Rubinstein, J., Fan, G.-C. Tsg101 positively regulates physiologic-like cardiac hypertrophy through FIP3-mediated endosomal recycling of IGF-1R.

Selective deletion of endothelial cell calpain in mice reduces diabetic cardiomyopathy by improving angiogenesis.

AIMS/HYPOTHESIS: The role of non-cardiomyocytes in diabetic cardiomyopathy has not been fully addressed. This study investigated whether endothelial cell calpain plays a role in myocardial endothelial injury and microvascular rarefaction in diabetes, thereby contributing to diabetic cardiomyopathy. METHODS: Endothelial cell-specific Capns1-knockout (KO) mice were generated. Conditions mimicking prediabetes and type 1 and type 2 diabetes were induced in these KO mice and their wild-type littermates. Myocardial function and coronary flow reserve were assessed by echocardiography. Histological analyses were performed to determine capillary density, cardiomyocyte size and fibrosis in the heart. Isolated aortas were assayed for neovascularisation. Cultured cardiac microvascular endothelial cells were stimulated with high palmitate. Angiogenesis and apoptosis were analysed. RESULTS: Endothelial cell-specific deletion of Capns1 disrupted calpain 1 and calpain 2 in endothelial cells, reduced cardiac fibrosis and hypertrophy, and alleviated myocardial dysfunction in mouse models of diabetes without significantly affecting systemic metabolic variables. These protective effects of calpain disruption in endothelial cells were associated with an increase in myocardial capillary density (wild-type vs Capns1-KO 3646.14 ± 423.51 vs 4708.7 ± 417.93 capillary number/high-power field in prediabetes, 2999.36 ± 854.77 vs 4579.22 ± 672.56 capillary number/high-power field in type 2 diabetes and 2364.87 ± 249.57 vs 3014.63 ± 215.46 capillary number/high-power field in type 1 diabetes) and coronary flow reserve. Ex vivo analysis of neovascularisation revealed more endothelial cell sprouts from aortic rings of prediabetic and diabetic Capns1-KO mice compared with their wild-type littermates. In cultured cardiac microvascular endothelial cells, inhibition of calpain improved angiogenesis and prevented apoptosis under metabolic stress. Mechanistically, deletion of Capns1 elevated the protein levels of ß-catenin in endothelial cells of Capns1-KO mice and constitutive activity of calpain 2 suppressed ß-catenin protein expression in cultured endothelial cells. Upregulation of ß-catenin promoted angiogenesis and inhibited apoptosis whereas knockdown of ß-catenin offset the protective effects of calpain inhibition in endothelial cells under metabolic stress. CONCLUSIONS/INTERPRETATION: These results delineate a primary role of calpain in inducing cardiac endothelial cell injury and impairing neovascularisation via suppression of ß-catenin, thereby promoting diabetic cardiomyopathy, and indicate that calpain is a promising therapeutic target to prevent diabetic cardiac complications.

Calpain-2 protects against heat stress-induced cardiomyocyte apoptosis and heart dysfunction by blocking p38 mitogen-activated protein kinase activation.

Cardiovascular dysfunction is a common complication among heatstroke patients, but its underlying mechanism is unclear. This study was designed to investigate the role of calpain-2 and its downstream signal pathway in heat stress-induced cardiomyocyte apoptosis and heart dysfunction. In cultured primary mouse neonatal cardiomyocytes (MNCs), heat stress (43°C for 2 hr) induced a heat-shock response, as indicated by upregulated heat-shock protein 27 (HSP27) expression and cellular apoptosis, as indicated by increased caspase-3 activity, DNA fragmentation and decreased cell viability. Meanwhile, heat stress decreased calpain activity, which was accompanied by downregulated calpain-2 expression and increased phosphorylation of p38, extraceIIuIar signaI-reguIated protein kinase (ERK1/2) and c-Jun N-terminaI kinase (JNK). Calpain-2 overexpression abrogated heat stress-induced apoptosis and phosphorylation of p38 and JNK, but not of ERK1/2. Blocking only p38 prevented heat stress-induced apoptosis in MNCs. In cardiac-specific calpain-2 overexpressing transgenic mice, p38 phosphorylation and cardiomyocyte apoptosis were decreased in the heart tissue of heatstroke mice, as revealed by western blot and terminal deoxynucleotidyl transferase dUTP nick end labelling assays, respectively. M-mode echocardiography also demonstrated that calpain-2 overexpression significantly improved heatstroke-induced decreases in ventricular end-diastolic volume and cardiac output. In conclusion, our study suggests that heat stress reduces calpain-2 expression, which then activates p38, leading to cardiomyocyte apoptosis and heart dysfunction.

Increased calpain-1 in mitochondria induces dilated heart failure in mice: role of mitochondrial superoxide anion.

We and others have reported that calpain-1 was increased in myocardial mitochondria from various animal models of heart disease. This study investigated whether constitutive up-regulation of calpain-1 restricted to mitochondria induced myocardial injury and heart failure and, if so, whether these phenotypes could be rescued by selective inhibition of mitochondrial superoxide production. Transgenic mice with human CAPN1 up-regulation restricted to mitochondria in cardiomyocytes (Tg-mtCapn1/tTA) were generated and characterized with low and high over-expression of transgenic human CAPN1 restricted to mitochondria, respectively. Transgenic up-regulation of mitochondria-targeted CAPN1 dose-dependently induced cardiac cell death, adverse myocardial remodeling, heart failure, and early death in mice, the changes of which were associated with mitochondrial dysfunction and mitochondrial superoxide generation. Importantly, a daily injection of mitochondria-targeted superoxide dismutase mimetics mito-TEMPO for 1 month starting from age 2 months attenuated cardiac cell death, adverse myocardial remodeling and heart failure, and reduced mortality in Tg-mtCapn1/tTA mice. In contrast, administration of TEMPO did not achieve similar cardiac protection in transgenic mice. Furthermore, transgenic up-regulation of mitochondria-targeted CAPN1 induced a reduction of ATP5A1 protein and ATP synthase activity in hearts. In cultured cardiomyocytes, increased calpain-1 in mitochondria promoted mitochondrial permeability transition pore (mPTP) opening and induced cell death, which were prevented by over-expression of ATP5A1, mito-TEMPO or cyclosporin A, an inhibitor of mPTP opening. In conclusion, this study has provided direct evidence demonstrating that increased mitochondrial calpain-1 is an important mechanism contributing to myocardial injury and heart failure by disrupting ATP synthase, and promoting mitochondrial superoxide generation and mPTP opening.

Administration of losartan preserves cardiomyocyte size and prevents myocardial dysfunction in tail-suspended mice by inhibiting p47phox phosphorylation, NADPH oxidase activation and MuRF1 expression.

BACKGROUND: Spaceflight or microgravity conditions cause myocardial atrophy and dysfunction, contributing to post-flight orthostatic intolerance. However, the underlying mechanisms remain incompletely understood and preventive approaches are limited. This study investigated whether and how losartan, a blocker of angiotensin-II receptor, preserved cardiomyocyte size and prevented myocardial dysfunction during microgravity. METHOD: Adult male mice were suspended with their tails to simulate microgravity. Echocardiography was performed to assess myocardial function. Heart weight and cardiomyocyte size were measured. NADPH oxidase activation was determined by analyzing membrane translocation of its cytosolic subunits including p47phox, p67phox and Rac1. Heart tissues were also assayed for oxidative stress, p47phox phosphorylation (Ser345), MuRF1 protein levels and angiotensin-II production. RESULTS: Tail-suspension for 28 days increased angiotensin-II production in hearts, decreased cardiomyocyte size and heart weight, and induced myocardial dysfunction. Administration of losartan preserved cardiomyocyte size and heart weight, and prevented myocardial dysfunction in tail-suspended mice. These cardioprotective effects of losartan were associated with inhibition of p47phox phosphorylation (Ser345), NADPH oxidase and oxidative stress in tail-suspended mouse hearts. Additionally, the NADPH oxidase inhibitor, apocynin, also reduced oxidative stress, preserved cardiomyocyte size and heart weight, and improved myocardial function in tail-suspended mice. Furthermore, losartan but not apocynin attenuated tail-suspension-induced up-regulation of MuRF1 protein in mouse hearts. CONCLUSIONS: Administration of losartan preserves cardiomyocyte size and prevents myocardial dysfunction under microgravity by blocking p47phox phosphorylation and NADPH oxidase activation, and by inhibiting MuRF1 expression. Thus, losartan may be a useful drug to prevent microgravity-induced myocardial abnormalities.

Mitochondrial ROS-induced lysosomal dysfunction impairs autophagic flux and contributes to M1 macrophage polarization in a diabetic condition.

Macrophage polarization toward the M1 phenotype and its subsequent inflammatory response have been implicated in the progression of diabetic complications. Despite adverse consequences of autophagy impairment on macrophage inflammation, the regulation of macrophage autophagy under hyperglycemic conditions is incompletely understood. Here, we report that the autophagy-lysosome system and mitochondrial function are impaired in streptozotocin (STZ)-induced diabetic mice and high glucose (HG)-stimulated RAW 264.7 cells. Mitochondrial dysfunction promotes reactive oxygen species (ROS) production and blocks autophagic flux by impairing lysosome function in macrophages under hyperglycemic conditions. Conversely, inhibition of mitochondrial ROS by Mito-TEMPO prevents HG-induced M1 macrophage polarization, and its effect is offset by blocking autophagic flux. The role of mitochondrial ROS in lysosome dysfunction and M1 macrophage polarization is also demonstrated in mitochondrial complex I defective RAW 264.7 cells induced by silencing NADH:ubiquinone oxidoreductase subunit-S4 (Ndufs4). These findings prove that mitochondrial ROS plays a key role in promoting macrophage polarization to inflammatory phenotype by impairing autophagy-lysosome system, which might provide clue to a novel treatment for diabetic complications.

Nicotinamide riboside promotes autolysosome clearance in preventing doxorubicin-induced cardiotoxicity.

Doxorubicin (DOX) is widely used as a first-line chemotherapeutic drug for various malignancies. However, DOX causes severe cardiotoxicity, which limits its clinical uses. Oxidative stress is one of major contributors to DOX-induced cardiotoxicity. While autophagic flux serves as an important defense mechanism against oxidative stress in cardiomyocytes, recent studies have demonstrated that DOX induces the blockage of autophagic flux, which contributes to DOX cardiotoxicity. The present study investigated whether nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD)+, prevents DOX cardiotoxicity by improving autophagic flux. We report that administration of NR elevated NAD+ levels, and reduced cardiac injury and myocardial dysfunction in DOX-injected mice. These protective effects of NR were recapitulated in cultured cardiomyocytes upon DOX treatment. Mechanistically, NR prevented the blockage of autophagic flux, accumulation of autolysosomes, and oxidative stress in DOX-treated cardiomyocytes, the effects of which were associated with restoration of lysosomal acidification. Furthermore, inhibition of lysosomal acidification or SIRT1 abrogated these protective effects of NR during DOX-induced cardiotoxicity. Collectively, our study shows that NR enhances autolysosome clearance via the NAD+/SIRT1 signaling, thereby preventing DOX-triggered cardiotoxicity.

Calpain-2 promotes MKP-1 expression protecting cardiomyocytes in both in vitro and in vivo mouse models of doxorubicin-induced cardiotoxicity.

We recently reported that doxorubicin decreased the expression of calpain-1/2, while inhibition of calpain activity promoted doxorubicin-induced cardiac injury in mice. In this study, we investigated whether and how elevation of calpain-2 could affect doxorubicin-triggered cardiac injury. Transgenic mice with inducible cardiomyocyte-specific expression of calpain-2 were generated. An acute cardiotoxicity was induced in both transgenic mice and their relevant wild-type littermates by injection of a single dose of doxorubicin (20 mg/kg) and cardiac injury was analyzed 5 days after doxorubicin injection. Cardiomyocyte-specific up-regulation of calpain-2 did not induce any adverse cardiac phenotypes under physiological conditions by age 3 months, but significantly reduced myocardial injury and improved myocardial function in doxorubicin-treated mice. Cardiac protection of calpain-2 up-regulation was also observed in a mouse model of chronic doxorubicin cardiotoxicity. Up-regulation of calpain-2 increased the protein levels of mitogen activated protein kinase phosphatase-1 (MKP-1) in cultured mouse cardiomyocytes and heart tissues. Over-expression of MKP-1 prevented, whereas knockdown of MKP-1 augmented doxorubicin-induced apoptosis in cultured cardiomyocytes. Moreover, knockdown of MKP-1 offset calpain-2-elicited protective effects against doxorubicin-induced injury in cultured cardiomyocytes. Mechanistically, up-regulation of calpain-2 reduced the protein levels of phosphatase and tensin homolog and consequently promoted Akt activation, leading to increased MKP-1 protein steady-state levels by inhibiting its degradation. Collectively, this study reveals a new role of calpain-2 in promoting MKP-1 expression via phosphatase and tensin homolog/Akt signaling. This study also suggests that calpain-2/MKP-1 signaling may represent new therapeutic targets for doxorubicin-induced cardiac injury.

MicroRNA-223-5p and -3p Cooperatively Suppress Necroptosis in Ischemic/Reperfused Hearts.

Recent studies have shown that myocardial ischemia/reperfusion (I/R)-induced necrosis can be controlled by multiple genes. In this study, we observed that both strands (5p and 3p) of miR-223 were remarkably dysregulated in mouse hearts upon I/R. Precursor miR-223 (pre-miR-223) transgenic mouse hearts exhibited better recovery of contractile performance over reperfusion period and lesser degree of myocardial necrosis than wild type hearts upon ex vivo and in vivo myocardial ischemia. Conversely, pre-miR-223 knock-out (KO) mouse hearts displayed opposite effects. Furthermore, we found that the RIP1/RIP3/MLKL necroptotic pathway and inflammatory response were suppressed in transgenic hearts, whereas they were activated in pre-miR-223 KO hearts upon I/R compared with wild type controls. Accordingly, treatment of pre-miR-223 KO mice with necrostatin-1s, a potent necroptosis inhibitor, significantly decreased I/R-triggered cardiac necroptosis, infarction size, and dysfunction. Mechanistically, we identified two critical cell death receptors, TNFR1 and DR6, as direct targets of miR-223-5p, whereas miR-223-3p directly suppressed the expression of NLRP3 and IκB kinase α, two important mediators known to be involved in I/R-induced inflammation and cell necroptosis. Our findings indicate that miR-223-5p/-3p duplex works together and cooperatively inhibits I/R-induced cardiac necroptosis at multiple layers. Thus, pre-miR-223 may constitute a new therapeutic agent for the treatment of ischemic heart disease.

Overexpression of miR-223 Tips the Balance of Pro- and Anti-hypertrophic Signaling Cascades toward Physiologic Cardiac Hypertrophy.

MicroRNAs (miRNAs) have been extensively examined in pathological cardiac hypertrophy. However, few studies focused on profiling the miRNA alterations in physiological hypertrophic hearts. In this study we generated a transgenic mouse model with cardiac-specific overexpression of miR-223. Our results showed that elevation of miR-223 caused physiological cardiac hypertrophy with enhanced cardiac function but no fibrosis. Using the next generation RNA sequencing, we observed that most of dys-regulated genes (e.g. Atf3/5, Egr1/3, Sfrp2, Itgb1, Ndrg4, Akip1, Postn, Rxfp1, and Egln3) in miR-223-transgenic hearts were associated with cell growth, but they were not directly targeted by miR-223. Interestingly, these dys-regulated genes are known to regulate the Akt signaling pathway. We further identified that miR-223 directly interacted with 3'-UTRs of FBXW7 and Acvr2a, two negative regulators of the Akt signaling. However, we also validated that miR-223 directly inhibited the expression of IGF-1R and ß1-integrin, two positive regulators of the Akt signaling. Lastly, Western blotting did reveal that Akt was activated in miR-223-overexpressing hearts. Adenovirus-mediated overexpression of miR-223 in neonatal rat cardiomyocytes induced cell hypertrophy, which was blocked by the addition of MK2206, a specific inhibitor of Akt Taken together, these data represent the first piece of work showing that miR-223 tips the balance of promotion and inactivation of Akt signaling cascades toward activation of Akt, a key regulator of physiological cardiac hypertrophy. Thus, our study suggests that the ultimate phenotype outcome of a miRNA may be decided by the secondary net effects of the whole target network rather than by several primary direct targets in an organ/tissue.