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BACKGROUND: Unlike conventional microsecond pulsed electrical fields that primarily target the cell membranes, nanosecond pulses are thought to primarily electroporate intracellular organelles. We conducted a comprehensive preclinical assessment of catheter-based endocardial nanosecond pulsed field ablation in swine. METHODS: A novel endocardial nanosecond pulsed field ablation system was evaluated in a total of 25 swine. Using either a low-dose (5-second duration) or high-dose (15-second duration) strategy, thoracic veins and discrete atrial and ventricular sites were ablated. Predetermined survival periods were <1 (n=1), ≈2 (n=7), ≈7 (n=6), 14 (n=2), or ≈28 (n=9) days, and venous isolation was assessed before euthanasia. Safety assessments included evaluation of esophageal effects, phrenic nerve function, and changes in venous caliber. All tissues were subject to careful gross pathological and histopathologic examination. RESULTS: All (100%) veins (13 low-dose, 34 high-dose) were acutely isolated, and all reassessed veins (6 low-dose, 15 highdose) were durably isolated. All examined vein lesions (10 low-dose, 22 high-dose) were transmural. Vein diameters (n=15) were not significantly changed. Of the animals assessed for phrenic palsy (n=9), 3 (33%) demonstrated only transient palsy. There were no differences between dosing strategies. Thirteen mitral isthmus lesions were analyzed, and all 13 (100%) were transmural (depth, 6.4±0.4 mm). Ventricular lesions were 14.7±4.5 mm wide and 7.1±1.3 mm deep, with high-dose lesions deeper than low-dose (7.9±1.2 versus 6.2±0.8 mm; P=0.007). The esophagus revealed nontransmural adventitial surface lesions in 5 of 5 (100%) animals euthanized early (2 days) post-ablation. In the 10 animals euthanized later (14–28 days), all animals demonstrated significant esophageal healing—8 with complete resolution, and 2 with only trace fibrosis. CONCLUSIONS: A novel, endocardial nanosecond pulsed field ablation system provides acute and durable venous isolation and linear lesions. Transient phrenic injury and nontransmural esophageal lesions can occur with worst-case assessments suggesting limits to pulsed field ablation tissue selectivity and the need for dedicated assessments during clinical studies. [ABSTRACT FROM AUTHOR]

Muscle-specific RING finger protein-1 (MuRF1) is an E3 ligase that inhibits cardiac hypertrophy. However, how MuRF1 regulates cardiac hypertrophy and function during pressure overload (PO) remains poorly understood. We investigated the role of endogenous MuRF1 in regulating cardiac hypertrophy in response to PO in vivo.Transverse aortic constriction (TAC) for 4 weeks significantly reduced expression of MuRF1 in the mouse heart. After 2 and 4 weeks of TAC, MuRF1 knockout (Murf1-/-) mice exhibited enhanced cardiac hypertrophy and left ventricular (LV) dysfunction compared with that of nontransgenic (NTg) mice. Histological analyses showed that Murf1-/- mice exhibited more severe fibrosis and apoptosis than NTg mice after TAC. TAC-induced increases in the activity of a nuclear factor of activated T cells (NFAT) luciferase reporter were significantly greater in Murf1-/- than in NTg mice. TAC-induced increases in calcineurin A (CnA) expression were also significantly enhanced in Murf1-/- compared with that in NTg mice. Coimmunoprecipitation assays showed that endogenous MuRF1 and CnA interact with one another. Polyubiquitination of CnA was attenuated in Murf1-/- mouse hearts at baseline and in response to TAC, and the protein stability of CnA was enhanced in cardiomyocytes, in which MuRF1 was downregulated in vitro. Furthermore, MuRF1 directly ubiquitinated CnA in vitro. Cardiac-specific overexpression of ZAKI-4β, an endogenous inhibitor of CnA, significantly suppressed the enhancement of TAC-induced cardiac hypertrophy and dysfunction, as well as increases in cardiac fibrosis and apoptosis, in Murf1-/- mice.Endogenous MuRF1 negatively regulates cardiac hypertrophy and dysfunction in response to PO through inhibition of the calcineurin-NFAT pathway. [ABSTRACT FROM AUTHOR]

The article investigates the impact of glycogen synthase kinase (GSK)-3ß on the efficiency of cardiac cell-based therapy (CBT) with mesenchymal stem cells (MSCs) following myocardial infarction (MI). Results reveal that GSK-3ß significantly enhances the efficiency of CBT with MSCs in the post-MI heart. The study reveals that GSK-3ß not just increases survival of MSCs, but likewise promotes cardiomyocyte differentiation and angiogenesis via Vegfa-dependent and -independent mechanisms.

The article examines the underlying signaling mechanism mediating autophagy during nutrient starvation in cardiomyocytes (CM). In particular, the study investigates the role of foxhead box, class O (FoxO) family of transcription factors, and its posttranslational modification in mediating starvation-induced autophagy. Results show that class III histone deacetylase Sirt1-induced increases in autophagic flux. Such result is said to play a critical role in maintaining left ventricular function during starvation.

The article presents a study that examined the functional role of stress-activated protein kinase (PKN) during myocardial ischemia and reperfusion (I/R). Research indicated that PKN is activated by hypotonic stress and I/R and that stimulation of PKN prevented cell death induced by hydrogen peroxide. It suggested that the cell-protective effect of PKN is cell-autonomous in cardiac myocytes and that its activation is an endogenous mechanism of cardioprotection. PKN mediated phosphorylation of alpha B crystalline and stimulation of proteasome activity.

Supplemental Digital Content is available in the text. Background: Takayasu arteritis (TAK) is an autoimmune systemic arteritis of unknown pathogenesis. Genome-wide association studies revealed that single-nucleotide polymorphisms in the MLX gene encoding the MLX (Max-like protein X) transcription factor are significantly associated with TAK in Japanese patients. MLX single-nucleotide polymorphism rs665268 is a missense mutation causing the Q139R substitution in the DNA-binding site of MLX. Methods: To elucidate the hypothesis that the single-nucleotide polymorphism of the MLX gene plays a critical role in the development of TAK, we conducted clinical and laboratory analyses. Results: We show that rs665268 significantly correlated with the severity of TAK, including the number of arterial lesions and morbidity of aortic regurgitation; the latter may be attributed to the fact that MLX mRNA expression was mostly detected in the aortic valve. Furthermore, the Q139R mutation caused structural changes in MLX, which resulted in enhanced formation of a heterodimer with MondoA, upregulation of TXNIP (thioredoxin-interacting protein) expression, and increase in the activity of the NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome and cellular oxidative stress. Furthermore, autophagy, which negatively regulates inflammasome activation, was suppressed by the Q139R mutation in MLX. The MLX-Q139R mutant significantly induced macrophage proliferation and macrophage–endothelium interaction, which was abolished by the treatment with SBI-477, an inhibitor of MondoA nuclear translocation. Our findings suggest that the Q139R substitution in MLX plays a crucial role in the pathogenesis of TAK. Conclusions: MLX-Q139R mutation plays a crucial role in the pathogenesis of TAK through promoting inflammasome formation. [ABSTRACT FROM AUTHOR]

Background: Takayasu arteritis (TAK) is an autoimmune systemic arteritis of unknown pathogenesis. Genome-wide association studies revealed that single-nucleotide polymorphisms in the MLX gene encoding the MLX (Max-like protein X) transcription factor are significantly associated with TAK in Japanese patients. MLX single-nucleotide polymorphism rs665268 is a missense mutation causing the Q139R substitution in the DNA-binding site of MLX., Methods: To elucidate the hypothesis that the single-nucleotide polymorphism of the MLX gene plays a critical role in the development of TAK, we conducted clinical and laboratory analyses., Results: We show that rs665268 significantly correlated with the severity of TAK, including the number of arterial lesions and morbidity of aortic regurgitation; the latter may be attributed to the fact that MLX mRNA expression was mostly detected in the aortic valve. Furthermore, the Q139R mutation caused structural changes in MLX, which resulted in enhanced formation of a heterodimer with MondoA, upregulation of TXNIP (thioredoxin-interacting protein) expression, and increase in the activity of the NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome and cellular oxidative stress. Furthermore, autophagy, which negatively regulates inflammasome activation, was suppressed by the Q139R mutation in MLX. The MLX-Q139R mutant significantly induced macrophage proliferation and macrophage-endothelium interaction, which was abolished by the treatment with SBI-477, an inhibitor of MondoA nuclear translocation. Our findings suggest that the Q139R substitution in MLX plays a crucial role in the pathogenesis of TAK., Conclusions: MLX-Q139R mutation plays a crucial role in the pathogenesis of TAK through promoting inflammasome formation.

Background: Mitochondrial autophagy is an important mediator of mitochondrial quality control in cardiomyocytes. The occurrence of mitochondrial autophagy and its significance during cardiac hypertrophy are not well understood., Methods and Results: Mice were subjected to transverse aortic constriction (TAC) and observed at multiple time points up to 30 days. Cardiac hypertrophy developed after 5 days, the ejection fraction was reduced after 14 days, and heart failure was observed 30 days after TAC. General autophagy was upregulated between 1 and 12 hours after TAC but was downregulated below physiological levels 5 days after TAC. Mitochondrial autophagy, evaluated by electron microscopy, mitochondrial content, and Keima with mitochondrial localization signal, was transiently activated at ≈3 to 7 days post-TAC, coinciding with mitochondrial translocation of Drp1. However, it was downregulated thereafter, followed by mitochondrial dysfunction. Haploinsufficiency of Drp1 abolished mitochondrial autophagy and exacerbated the development of both mitochondrial dysfunction and heart failure after TAC. Injection of Tat-Beclin 1, a potent inducer of autophagy, but not control peptide, on day 7 after TAC, partially rescued mitochondrial autophagy and attenuated mitochondrial dysfunction and heart failure induced by overload. Haploinsufficiency of either drp1 or beclin 1 prevented the rescue by Tat-Beclin 1, suggesting that its effect is mediated in part through autophagy, including mitochondrial autophagy., Conclusions: Mitochondrial autophagy is transiently activated and then downregulated in the mouse heart in response to pressure overload. Downregulation of mitochondrial autophagy plays an important role in mediating the development of mitochondrial dysfunction and heart failure, whereas restoration of mitochondrial autophagy attenuates dysfunction in the heart during pressure overload., (© 2016 American Heart Association, Inc.)

Rationale: Both fusion and fission contribute to mitochondrial quality control. How unopposed fusion affects survival of cardiomyocytes and left ventricular function in the heart is poorly understood., Objective: We investigated the role of dynamin-related protein 1 (Drp1), a GTPase that mediates mitochondrial fission, in mediating mitochondrial autophagy, ventricular function, and stress resistance in the heart., Methods and Results: Drp1 downregulation induced mitochondrial elongation, accumulation of damaged mitochondria, and increased apoptosis in cardiomyocytes at baseline. Drp1 downregulation also suppressed autophagosome formation and autophagic flux at baseline and in response to glucose deprivation in cardiomyocytes. The lack of lysosomal translocation of mitochondrially targeted Keima indicates that Drp1 downregulation suppressed mitochondrial autophagy. Mitochondrial elongation and accumulation of damaged mitochondria were also observed in tamoxifen-inducible cardiac-specific Drp1 knockout mice. After Drp1 downregulation, cardiac-specific Drp1 knockout mice developed left ventricular dysfunction, preceded by mitochondrial dysfunction, and died within 13 weeks. Autophagic flux is significantly suppressed in cardiac-specific Drp1 knockout mice. Although left ventricular function in cardiac-specific Drp1 heterozygous knockout mice was normal at 12 weeks of age, left ventricular function decreased more severely after 48 hours of fasting, and the infarct size/area at risk after ischemia/reperfusion was significantly greater in cardiac-specific Drp1 heterozygous knockout than in control mice., Conclusions: Disruption of Drp1 induces mitochondrial elongation, inhibits mitochondrial autophagy, and causes mitochondrial dysfunction, thereby promoting cardiac dysfunction and increased susceptibility to ischemia/reperfusion., (© 2014 American Heart Association, Inc.)

Rationale: Overexpression of muscle atrophy F-box (MAFbx/atrogin-1), an E3 ubiquitin ligase, induces proteasomal degradation in cardiomyocytes. The role of endogenous MAFbx in regulating cardiac hypertrophy and failure remains unclear., Objective: We investigated the role of MAFbx in regulating cardiac hypertrophy and function in response to pressure overload. Transverse aortic constriction (TAC) was applied to MAFbx knockout (KO) and wild-type (WT) mice., Methods and Results: Expression of MAFbx in WT mice was significantly increased by TAC. TAC-induced increases in cardiac hypertrophy were significantly smaller in MAFbx KO than in WT mice. There was significantly less lung congestion and interstitial fibrosis in MAFbx KO than in WT mice. MAFbx KO also inhibited β-adrenergic cardiac hypertrophy. DNA microarray analysis revealed that activation of genes associated with the transcription factor binding site for the nuclear factor-κB family were inhibited in MAFbx KO mice compared with WT mice after TAC. Although the levels of IκB-α were significantly decreased after TAC in WT mice, they were increased in MAFbx KO mice. MAFbx regulates ubiquitination and proteasomal degradation of IκB-α in cardiomyocytes. In primary cultured rat cardiomyocytes, phenylephrine-induced activation of nuclear factor-κB and hypertrophy were significantly suppressed by MAFbx knockdown but were partially rescued by overexpression of nuclear factor-κB p65., Conclusions: MAFbx plays an essential role in mediating cardiac hypertrophy in response to pressure overload. Downregulation of MAFbx inhibits cardiac hypertrophy in part through stabilization of IκB-α and inactivation of nuclear factor-κB. Taken together, inhibition of MAFbx attenuates pathological hypertrophy, thereby protecting the heart from progression into heart failure.

Rationale: Glycogen synthase kinase (GSK)-3β upregulates cardiac genes in bone marrow-derived mesenchymal stem cells (MSCs) in vitro. Ex vivo modification of signaling mechanisms in MSCs may improve the efficiency of cardiac cell-based therapy (CBT)., Objective: To test the effect of GSK-3β on the efficiency of CBT with MSCs after myocardial infarction (MI)., Methods and Results: MSCs overexpressing either GSK-3β (GSK-3β-MSCs), LacZ (LacZ-MSCs), or saline was injected into the heart after coronary ligation. A significant improvement in the mortality and left ventricular (LV) function was observed at 12 weeks in GSK-3β-MSC-injected mice compared with in LacZ-MSC- or saline-injected mice. MI size and LV remodeling were reduced in GSK-3β-MSC-injected mice compared with in LacZ-MSC- or saline-injected ones. GSK-3β increased survival and increased cardiomyocyte differentiation of MSCs, as evidenced by activation of an Nkx2.5-LacZ reporter and upregulation of troponin T. Injection of GSK-3β-MSCs induced Ki67-positive myocytes and c-Kit-positive cells, suggesting that GSK-3β-MSCs upregulate cardiac progenitor cells. GSK-3β-MSCs also increased capillary density and upregulated paracrine factors, including vascular endothelial growth factor A (Vegfa). Injection of GSK-3β-MSCs in which Vegfa had been knocked down abolished the increase in survival and capillary density. However, the decrease in MI size and LV remodeling and the improvement of LV function were still observed in MI mice injected with GSK-3β-MSCs without Vegfa., Conclusions: GSK-3β significantly improves the efficiency of CBT with MSCs in the post-MI heart. GSK-3β not only increases survival of MSCs but also induces cardiomyocyte differentiation and angiogenesis through Vegfa-dependent and -independent mechanisms.

Background: Silent information regulator 1 (Sirt1), a class III histone deacetylase, retards aging and protects the heart from oxidative stress. We here examined whether Sirt1 is protective against myocardial ischemia/reperfusion (I/R)., Methods and Results: Protein and mRNA expression of Sirt1 is significantly reduced by I/R. Cardiac-specific Sirt1(-/-) mice exhibited a significant increase (44±5% versus 15±5%; P=0.01) in the size of myocardial infarction/area at risk. In transgenic mice with cardiac-specific overexpression of Sirt1, both myocardial infarction/area at risk (15±4% versus 36±8%; P=0.004) and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive nuclei (4±3% versus 10±1%; P<0.003) were significantly reduced compared with nontransgenic mice. In Langendorff-perfused hearts, the functional recovery during reperfusion was significantly greater in transgenic mice with cardiac-specific overexpression of Sirt1 than in nontransgenic mice. Sirt1 positively regulates expression of prosurvival molecules, including manganese superoxide dismutase, thioredoxin-1, and Bcl-xL, whereas it negatively regulates the proapoptotic molecules Bax and cleaved caspase-3. The level of oxidative stress after I/R, as evaluated by anti-8-hydroxydeoxyguanosine staining, was negatively regulated by Sirt1. Sirt1 stimulates the transcriptional activity of FoxO1, which in turn plays an essential role in mediating Sirt1-induced upregulation of manganese superoxide dismutase and suppression of oxidative stress in cardiac myocytes. Sirt1 plays an important role in mediating I/R-induced increases in the nuclear localization of FoxO1 in vivo., Conclusions: These results suggest that Sirt1 protects the heart from I/R injury through upregulation of antioxidants and downregulation of proapoptotic molecules through activation of FoxO and decreases in oxidative stress.

Objective: Proliferation and migration of smooth muscle cells (SMCs) and migration and accumulation of monocytes and T cells are landmark events in the development of arteriosclerosis. SMC proliferation in the intima induces interruption of blood flow and results in ischemia and graft rejection. Inducible costimulator (ICOS) is a major costimulator of T cell activation. However, the effect of costimulatory molecules on the formation of neointimal hyperplasia has not been fully elucidated. We examined the role of the ICOS pathway in SMC proliferation., Methods and Results: ICOS ligand (ICOSL) was detected in SMCs stimulated by interleukin (IL)-1beta, and coculture of stimulated SMCs and activated T cells induced SMC proliferation. Inhibition of the ICOS pathway resulted in inhibition of SMC proliferation. In models of transplantation and vascular injury, ICOSL was induced in SMCs in the neointima. Expression of IL-1beta, a key inducer of ICOSL expression, was significantly reduced in mice treated with anti-ICOS antibody or soluble form of ICOS (ICOSIg) and in ICOS-deficient mice. Inhibition of the ICOS pathway significantly suppressed neointimal thickening., Conclusions: These results indicate that ICOS on activated T cells contributes to neointimal formation through the regulation of SMC proliferation. These findings provide insights into new therapeutic strategies for arteriosclerosis.

Background: Peroxisome proliferator-activated receptor-gamma plays an important role in regulating inflammation. Although cardiac transplantation is an established therapy for patients with end-stage heart disease, allograft rejection is a major concern for long-term survival. We investigated the role of pioglitazone in acute and chronic rejection in a murine cardiac transplantation model., Methods and Results: We performed heterotopic murine cardiac transplantation in total allomismatch or major histocompatibility complex class II-mismatched combinations. Recipient mice were given standard chow or chow containing pioglitazone (3 mg.kg(-1).d(-1)) beginning 1 day before cardiac transplantation. In acute rejection, animals given pioglitazone showed significantly longer cardiac allograft survival than control mice (mean survival time, 34.6+/-7.8 versus 8.4+/-0.4 days; P<0.003). Treatment with pioglitazone significantly suppressed graft expression of interferon-gamma and monocyte chemoattractant protein-1. In chronic rejection, neointimal hyperplasia was significantly lower in allografts from mice treated with pioglitazone (luminal occlusion, 25.1+/-8.8%) than in those from control mice (65.8+/-7.3%, P<0.001). Pioglitazone-treated allografts showed significantly reduced expression of interferon-gamma, interleukin-10, and monocyte chemoattractant protein-1. We performed mixed lymphocyte reactions and in vitro proliferation assays of smooth muscle cells. Addition of pioglitazone to mixed lymphocyte reactions inhibited proliferation of T cells. Smooth muscle cells showed significant proliferation when cocultured with activated splenocytes. This proliferation was significantly inhibited by the addition of pioglitazone (1 micromol/L)., Conclusions: Pioglitazone prolongs allograft survival and attenuates neointimal hyperplasia through the suppression of proliferation of smooth muscle cells. Pioglitazone may be a novel means to prevent acute and chronic allograft rejection.

Background: Programmed death-1 (PD-1), a member of the CD28 family, has been identified. PD-1 is involved in the negative regulation of some immune responses. We evaluated the role of PD-ligand 1 (PD-L1) in graft arterial disease (GAD) of cardiac allografts and in smooth muscle cells (SMCs)., Methods and Results: C57BL/6 murine hearts were transplanted into B6.C-H2KhEg mice for examination of GAD. PD-L1 was expressed in SMCs of the thickened intima in the graft coronary arteries, and administration of anti-PD-L1 monoclonal antibody (mAb) enhanced the progression of GAD (luminal occlusion: 55+/-5.0% versus 9.8+/-4.3%, P<0.05). The expressions of interferon gamma (IFN-gamma) and tumor necrosis factor alpha of cardiac allografts were upregulated in response to anti-PD-L1 mAb treatment. In vitro, PD-L1 expression was induced in SMCs in response to IFN-gamma stimulation. Sensitized splenocytes increased SMC proliferation, and anti-PD-L1 mAb in combination with IFN-gamma stimulation increased this proliferation., Conclusions: The PD-L1 pathway regulates both the proliferation of SMCs and GAD. Thus, control of this interaction is a promising strategy for suppression of GAD.