Nitroxide-enhanced MRI of cardiovascular oxidative stress.

BACKGROUND: In vivo imaging of oxidative stress can facilitate the understanding and treatment of cardiovascular diseases. We evaluated nitroxide-enhanced MRI with 3-carbamoyl-proxyl (3CP) for the detection of myocardial oxidative stress. METHODS: Three mouse models of cardiac oxidative stress were imaged, namely angiotensin II (Ang II) infusion, myocardial infarction (MI), and high-fat high-sucrose (HFHS) diet-induced obesity (DIO). For the Ang II model, mice underwent MRI at baseline and after 7 days of Ang II (n = 8) or saline infusion (n = 8). For the MI model, mice underwent MRI at baseline (n = 10) and at 1 (n = 8), 4 (n = 9), and 21 (n = 8) days after MI. For the HFHS-DIO model, mice underwent MRI at baseline (n = 20) and 18 weeks (n = 13) after diet initiation. The 3CP reduction rate, Kred , computed using a tracer kinetic model, was used as a metric of oxidative stress. Dihydroethidium (DHE) staining of tissue sections was performed on Day 1 after MI. RESULTS: For the Ang II model, Kred was higher after 7 days of Ang II versus other groups (p < 0.05). For the MI model, Kred , in the infarct region was significantly elevated on Days 1 and 4 after MI (p < 0.05), whereas Kred in the noninfarcted region did not change after MI. DHE confirmed elevated oxidative stress in the infarct zone on Day 1 after MI. After 18 weeks of HFHS diet, Kred was higher in mice after diet versus baseline (p < 0.05). CONCLUSIONS: Nitroxide-enhanced MRI noninvasively quantifies tissue oxidative stress as one component of a multiparametric preclinical MRI examination. These methods may facilitate investigations of oxidative stress in cardiovascular disease and related therapies.

Implications of scar structure and mechanics for post-infarction cardiac repair and regeneration.

Regenerating cardiac muscle lost during a heart attack is a topic of broad interest and enormous potential impact. One promising approach is to regenerate or re-engineer new myocardium in situ, at the site of damage, by injecting cells, growth factors, and other materials, or by reprogramming aspects of the normal wound healing process. A wide variety of strategies have been explored, from promoting angiogenesis to injection of a variety of different progenitor cell types, to re-engineering resident cells to produce key growth factors or even transdifferentiate into myocytes. Despite substantial progress and continued promise, clinical impact of this work has fallen short of expectations. One contributing factor may be that many efforts focus primarily on generating cardiomyocytes, with less attention to re-engineering the extracellular matrix (ECM). Yet the role of the ECM is particularly crucial to consider following myocardial infarction, which leads to rapid formation of a collagen-rich scar. This review combines a brief summary of current efforts to regenerate cardiomyocytes with what is currently known about the structure and mechanics of post-infarction scar, with the goal of identifying principles that can guide efforts to produce new myocytes embedded in an extracellular environment that facilitates their differentiation, maintenance, and function.

Klf4 has an unexpected protective role in perivascular cells within the microvasculature.

Recent smooth muscle cell (SMC) lineage-tracing studies have revealed that SMCs undergo remarkable changes in phenotype during development of atherosclerosis. Of major interest, we demonstrated that Kruppel-like factor 4 (KLF4) in SMCs is detrimental for overall lesion pathogenesis, in that SMC-specific conditional knockout of the KLF4 gene ( Klf4) resulted in smaller, more-stable lesions that exhibited marked reductions in the numbers of SMC-derived macrophage- and mesenchymal stem cell-like cells. However, since the clinical consequences of atherosclerosis typically occur well after our reproductive years, we sought to identify beneficial KLF4-dependent SMC functions that were likely to be evolutionarily conserved. We tested the hypothesis that KLF4-dependent SMC transitions play an important role in the tissue injury-repair process. Using SMC-specific lineage-tracing mice positive and negative for simultaneous SMC-specific conditional knockout of Klf4, we demonstrate that SMCs in the remodeling heart after ischemia-reperfusion injury (IRI) express KLF4 and transition to a KLF4-dependent macrophage-like state and a KLF4-independent myofibroblast-like state. Moreover, heart failure after IRI was exacerbated in SMC Klf4 knockout mice. Surprisingly, we observed a significant cardiac dilation in SMC Klf4 knockout mice before IRI as well as a reduction in peripheral resistance. KLF4 chromatin immunoprecipitation-sequencing analysis on mesenteric vascular beds identified potential baseline SMC KLF4 target genes in numerous pathways, including PDGF and FGF. Moreover, microvascular tissue beds in SMC Klf4 knockout mice had gaps in lineage-traced SMC coverage along the resistance arteries and exhibited increased permeability. Together, these results provide novel evidence that Klf4 has a critical maintenance role within microvascular SMCs: it is required for normal SMC function and coverage of resistance arteries. NEW & NOTEWORTHY We report novel evidence that the Kruppel-like factor 4 gene ( Klf4) has a critical maintenance role within microvascular smooth muscle cells (SMCs). SMC-specific Klf4 knockout at baseline resulted in a loss of lineage-traced SMC coverage of resistance arteries, dilation of resistance arteries, increased blood flow, and cardiac dilation.

Neuraminidase-mediated desialylation augments AAV9-mediated gene expression in skeletal muscle.

BACKGROUND: Following systemic delivery, AAV9-mediated gene expression is significantly increased in ischemic versus non-ischemic muscle, suggesting that AAV9 is an attractive vector for treating peripheral arterial disease. Potential mechanisms underlying ischemia-augmented expression include: (i) increased vascular permeability and (ii) "unmasking" of endogenous AAV9 receptors. In the present study, we aimed to reconstitute the ischemic induction of AAV9 in vivo, using local injection of histamine (to increase vascular permeability) and neuraminidase (to desialylate cell surface glycans). METHODS: Bioassays were performed to optimize the effects of histamine and neuraminidase after intramuscular injection. Histamine and/or neuraminidase were then injected intramuscularly shortly before intravenous injection of an AAV9 vector expressing luciferase. Luciferase expression was serially assessed with bioluminescence imaging. At the end of the study, tissues were harvested for assays of luciferase activity and AAV9 genome copy number aiming to assess AAV-mediated gene expression and transduction, respectively. RESULTS: Intramuscular injection of either neuraminidase or neuraminidase plus histamine significantly increased both transduction and gene expression, whereas histamine alone had little effect. Pre-injection with neuraminidase increased AAV9-mediated gene delivery by four- to nine-fold and luciferase activity by 60-100-fold. Luciferase activity in neuraminidase-injected muscle was > 100-fold higher than in any off-target tissue (including heart, liver and brain). CONCLUSIONS: The ischemic induction of AAV9-mediated gene expression in muscle can largely be reconstituted by pre-injecting neuraminidase intranmuscularly. This strategy may prove useful in future human gene therapy protocols as a quick and efficient means to selectively target systemically injected AAV9 to localized regions of muscle, thus decreasing the potential for adverse effects in off-target tissues.

Chemical Endoplasmic Reticulum Chaperone Alleviates Doxorubicin-Induced Cardiac Dysfunction.

RATIONALE: Doxorubicin is an effective chemotherapeutic agent for cancer, but its use is often limited by cardiotoxicity. Doxorubicin causes endoplasmic reticulum (ER) dilation in cardiomyocytes, and we have demonstrated that ER stress plays important roles in the pathophysiology of heart failure. OBJECTIVE: We evaluated the role of ER stress in doxorubicin-induced cardiotoxicity and examined whether the chemical ER chaperone could prevent doxorubicin-induced cardiac dysfunction. METHODS AND RESULTS: We confirmed that doxorubicin caused ER dilation in mouse hearts, indicating that doxorubicin may affect ER function. Doxorubicin activated an ER transmembrane stress sensor, activating transcription factor 6, in cultured cardiomyocytes and mouse hearts. However, doxorubicin suppressed the expression of genes downstream of activating transcription factor 6, including X-box binding protein 1. The decreased levels of X-box binding protein 1 resulted in a failure to induce the expression of the ER chaperone glucose-regulated protein 78 which plays a major role in adaptive responses to ER stress. In addition, doxorubicin activated caspase-12, an ER membrane-resident apoptotic molecule, which can lead to cardiomyocyte apoptosis and cardiac dysfunction. Cardiac-specific overexpression of glucose-regulated protein 78 by adeno-associated virus 9 or the administration of the chemical ER chaperone 4-phenylbutyrate attenuated caspase-12 cleavage, and alleviated cardiac apoptosis and dysfunction induced by doxorubicin. CONCLUSIONS: Doxorubicin activated the ER stress-initiated apoptotic response without inducing the ER chaperone glucose-regulated protein 78, further augmenting ER stress in mouse hearts. Cardiac-specific overexpression of glucose-regulated protein 78 or the administration of the chemical ER chaperone alleviated the cardiac dysfunction induced by doxorubicin and may facilitate the safe use of doxorubicin for cancer treatment.

Infarct-Sparing Effect of Adenosine A2B Receptor Agonist Is Primarily Due to Its Action on Splenic Leukocytes Via a PI3K/Akt/IL-10 Pathway.

BACKGROUND: Adenosine A2B receptor (A2BAR) agonist reduces myocardial reperfusion injury by acting on inflammatory cells. Recently, a cardiosplenic axis was shown to mediate the myocardial postischemic reperfusion injury. This study aimed to explore whether the infarct-squaring effect of A2BAR agonist was primarily due to its action on splenic leukocytes. METHODS: C57BL6 (wild type [WT]) mice underwent 40 min of left coronary artery occlusion followed by 60 min of reperfusion. A2BAR knockout (KO) and interleukin (IL)-10KO mice served as donors for splenic leukocytes. Acute splenectomy was performed 30 min before ischemia. The acute splenic leukocyte adoptive transfer was performed by injecting 5 × 106 live splenic leukocytes into splenectomized mice. BAY 60-6583, an A2BAR agonist, was injected by i.v. 15 min before ischemia. The infarct size (IS) was determined using 2,3,5-triphenyltetrazolium chloride and Phthalo blue staining. The expression of p-Akt and IL-10 was estimated by Western blotting. Immunofluorescence staining assessed the localization of IL-10 expression. RESULTS: BAY 60-6583 reduced the myocardial IS in intact mice but failed to reduce the same in splenectomized mice, which had a smaller IS than intact mice. BAY 60-6583 reduced the IS in splenectomized mice with the acute transfer of WT splenic leukocytes; however, it did not protect the heart of splenectomized mice with the acute transfer of A2BRKO splenic leukocytes. Furthermore, BAY 60-6583 increased the levels of p-Akt and IL-10 in the WT spleen. Moreover, it did not exert any protective effect in IL-10KO mice. CONCLUSIONS: A2BAR activation before ischemia stimulated the IL-10 production in splenic leukocytes via a PI3K/Akt pathway, thereby exerting anti-inflammatory effects that limited the myocardial reperfusion injury.

Stimulation of the Beta2 Adrenergic Receptor at Reperfusion Limits Myocardial Reperfusion Injury via an Interleukin-10-Dependent Anti-Inflammatory Pathway in the Spleen.

BACKGROUND: In addition to the airway-relaxing effects, ß2 adrenergic receptor (ß2AR) agonists are also found to have broad anti-inflammatory effects. The current study was conducted to define the role of ß2AR agonists in limiting myocardial ischemia/reperfusion injury (IRI). Methods and Results: Adult male wild-type (WT) and interleukin (IL)-10 knockout (KO) mice underwent a 40-min left coronary artery ligation and 60-min reperfusion. A selective ß2AR agonist, Clenbuterol, at doses of 0.1 µg or 1 µg/g weight i.v. 5 min before reperfusion, significantly reduced myocardial infarct size (IS) by 28% and 39% (vs. control, P<0.05) in WT mice respectively, but had no protective effect in IL-10 KO mice. Inhalational therapy with nebulized Clenbuterol, Albuterol, Salmeterol or Arformoterol immediately before ischemia significantly reduced IS (P<0.05) in WT mice. Splenectomy similarly reduced IS as Clenbuterol-treated mice, but intravenous Clenbuterol did not further reduce IS in splenectomized mice. In splenectomized WT mice, acute transfer of isolated splenocytes, not the Clenbuterol-pretreated splenocytes, restored the myocardial IS to the level of intact mice. Intravenous Clenbuterol significantly increased splenic protein levels of ß2AR, phosphorylated Akt and IL-10 and plasma IL-10, and inhibited the expression of pro-inflammatory mRNAs. CONCLUSIONS: Both intravenous and inhalational ß2AR agonists exert a cardioprotective effect against IRI by activating the anti-inflammatory ß2AR-IL-10 pathway.

Evaluation of pharmacokinetic and pharmacodynamic profiles of liposomes for the cell type-specific delivery of small molecule drugs.

Liposome-based drug formulations represent an exciting avenue of research as they increase efficacy to toxicity ratios. Current formulations rely on passive accumulation to the disease site where drug is taken up by the cells. Ligand mediated targeting increases the net accumulation of liposomes, however, an unexplored benefit is to potentially refine pharmacodynamics (PD) of a drug specifically to different cell types within diseased tissue. As a model system, we engineered cardiomyocyte- (I-1) and endothelial-targeted (B-40) liposomes to carry a VEGFR2 inhibitor (PTK787), and examined the effect of cell type-specific delivery on both pharmacokinetics (PK) and PD. Neovascularization in post-myocardial infarction was significantly reduced by B-40 liposomes loaded with PTK787 as compared to animals injected with I-1 liposomes, and profoundly more as compared to free PTK787. This study thus shows that the intraorgan targeting of drugs through cell type-specific delivery holds substantial promise towards lowering the minimal efficacious dose administered systemically.

The spleen contributes importantly to myocardial infarct exacerbation during post-ischemic reperfusion in mice via signaling between cardiac HMGB1 and splenic RAGE.

The spleen plays a critical role in post-infarct myocardial remodeling. However, the role of the spleen in exacerbating myocardial infarction (MI) during acute ischemia/reperfusion (I/R) injury is unknown. The present study tests the hypothesis that splenic leukocytes are activated by substances released from ischemic myocardium to subsequently exacerbate myocardial injury during reperfusion. The left coronary artery in C57BL/6 mice underwent various durations of occlusion followed by 60 min of reperfusion (denoted as min/min of I/R) with or without splenectomy prior to I/R injury. Splenectomy significantly decreased myocardial infarct size (IS) in 40'/60' and 50'/60' groups (p < 0.05); however, it had no effect on IS in 10'/60', 20'/60' and 30'/60' groups (p = NS). In the 20'/60' group, infusion of 40-min ischemic heart homogenate (40-IHH) upon reperfusion increased IS by >threefold versus infusion of 10-IHH (p < 0.05). Splenectomy abolished the infarct-exacerbating effect of 40-IHH, which was restored by splenic leukocyte adoptive transfer (SPAT). Furthermore, depletion of HMGB1 in the 40-IHH group abolished its infarct-exacerbating effect (p < 0.05), and 40-IHH failed to increase IS in both RAGE(-/-) mice and splenectomized wild-type mice with SPAT from RAGE(-/-) mice. The injection of 40-IHH significantly increased formyl peptide receptor 1 (FPR1) expression in sham spleens when compared to 10-IHH-treated sham and control mice. cFLFLF, a specific FPR1 antagonist, reduced myocardial neutrophil infiltration and abrogated the infarct-exacerbating effect of 40-IHH during reperfusion. A cardio (HMGB1)-splenic (RAGE receptor) signaling axis exists and contributes to myocardial infarct exacerbation during reperfusion after prolonged ischemic insults by activating splenic leukocytes. The FPR1 is a potential therapeutic target for inhibiting the cardio-splenic axis that augments infarct size during post-ischemic reperfusion.

Repeatability and variability of myocardial perfusion imaging techniques in mice: Comparison of arterial spin labeling and first-pass contrast-enhanced MRI.

PURPOSE: Preclinical imaging of myocardial blood flow (MBF) can elucidate molecular mechanisms underlying cardiovascular disease. We compared the repeatability and variability of two methods, first-pass MRI and arterial spin labeling (ASL), for imaging MBF in mice. METHODS: Quantitative perfusion MRI in mice was performed using both methods at rest, with a vasodilator, and one day after myocardial infarction. Image quality (score of 1-5; 5 best), between-session coefficient of variability (CVbs ), intra-user coefficient of variability (CVintra-user ), and inter-user coefficient of variability (CVinter-user ) were assessed. Acquisition time was 1-2 min for first-pass MRI and approximately 40 min for ASL. RESULTS: Image quality was higher for ASL (3.94 ± 0.09 versus 2.88 ± 0.10; P < 0.05). Infarct zone CVbs was lower with first-pass (17 ± 3% versus 46 ± 9%; P < 0.05). The stress perfusion CVintra-user was lower for ASL (3 ± 1% versus 14 ± 3%; P < 0.05). The stress perfusion CVinter-user was lower for ASL (4 ± 1% versus 17 ± 4%; P < 0.05). CONCLUSION: For low MBF conditions such as infarct, first-pass MRI is preferred due to better repeatability and variability. At high MBF such as at vasodilation, ASL may be more suitable due to superior image quality and lower user variability. First-pass MRI has a substantial speed advantage. Magn Reson Med 75:2394-2405, 2016. © 2015 Wiley Periodicals, Inc.

Splenic leukocytes mediate the hyperglycemic exacerbation of myocardial infarct size in mice.

Acute hyperglycemia during acute myocardial infarction is associated with worse myocardial injury and increased mortality. Using a mouse model of myocardial ischemia/reperfusion injury, we tested the hypothesis that acute hyperglycemia activates splenic leukocytes and subsequently exacerbates myocardial infarct size. We then examined whether the adverse effects of hyperglycemia could be attenuated by a potent anti-inflammatory agent (an agonist of the adenosine A2A receptor) administered immediately prior to reperfusion. C57BL6 (WT) mice underwent 30-min LAD occlusion and 60-min reperfusion with or without prior splenectomy. Acute hyperglycemia before ischemia increased myocardial infarct size (IS) by 43% (p < 0.05). Splenectomy before ischemia did not change IS (vs. control, p = NS) but did serve to prevent the exacerbation of IS by hyperglycemia. Acute hyperglycemia activated splenic leukocytes by increasing formyl peptide receptor expression and reactive oxygen species production before ischemia, and enhanced splenic neutrophil release with resultant peripheral neutrophilia and increased myocardial neutrophil infiltration during reperfusion. Acute adoptive transfer of splenic leukocytes to splenectomized mice before ischemia restored the hyperglycemic exacerbation of infarct size. ATL146e, an adenosine 2A receptor (A2AR) agonist, abolished neutrophilia during reperfusion and reduced IS in hyperglycemic mice. ATL146e also reduced IS in splenectomized hyperglycemic mice with transfer of WT splenic leukocytes, but not with transfer of splenic leukocytes from A2AR knockout mice. Acute hyperglycemia prior to myocardial ischemia and reperfusion exacerbates IS by activating splenic leukocytes. ATL146e administered at reperfusion suffices to abrogate the hyperglycemic exacerbation of IS by acting on A2ARs on splenic leukocytes.

The infarct-sparing effect of IB-MECA against myocardial ischemia/reperfusion injury in mice is mediated by sequential activation of adenosine A3 and A 2A receptors.

Conflicting results exist regarding the role of A3 adenosine receptors (A3ARs) in mediating cardioprotection during reperfusion following myocardial infarction. We hypothesized that the effects of the A3AR agonist IB-MECA to produce cardioprotection might involve activation of other adenosine receptor subtypes. C57Bl/6 (B6), A3AR KO, A2AAR KO, and A2AAR KO/WT bone marrow chimeric mice were assigned to 12 groups undergoing either hemodynamic studies or 45 min of LAD occlusion and 60 min of reperfusion. IB-MECA (100 µg/kg) or vehicle was administered by iv bolus 5 min before reperfusion. Radioligand binding assays showed that IB-MECA has high affinity for the mouse A3AR (K i = 0.17 ± 0.05 nM), but also can bind with lower affinity to the A1AR (9.0 ± 2.4 nM) or the A2AAR (56.5 ± 10.2 nM). IB-MECA caused bi-phasic hemodynamic changes, which were completely absent in A3AR KO mice and were modified by A2AAR blockade or deletion. IB-MECA stimulated histamine release, increased heart rate, and significantly reduced IF size in B6 mice from 61.5 ± 1.4 to 48.6 ± 2.4% of risk region (RR; 21% reduction, p < 0.05) but not in A3AR KO mice. Compared to B6, A3AR KO mice had significantly reduced IF size (p < 0.05). In B6/B6 bone marrow chimeras, IB-MECA caused a 47% reduction of IF size (from 47.3 ± 3.9 to 24.7 ± 4.5, p < 0.05). However, no significant cardioprotective effect of IB-MECA was observed in A2AARKO/B6 mice, which lacked A2AARs only on their bone marrow-derived cells. Activation of A3ARs induces a bi-phasic hemodynamic response, which is partially mediated by activation of A2AARs. The cardioprotective effect of IB-MECA is due to the initial activation of A3AR followed by activation of A2AARs in bone marrow-derived cells.

Obesity-Induced Coronary Microvascular Disease Is Prevented by iNOS Deletion and Reversed by iNOS Inhibition.

Coronary microvascular disease (CMD) caused by obesity and diabetes is major contributor to heart failure with preserved ejection fraction; however, the mechanisms underlying CMD are not well understood. Using cardiac magnetic resonance applied to mice fed a high-fat, high-sucrose diet as a model of CMD, we elucidated the role of inducible nitric oxide synthase (iNOS) and 1400W, an iNOS antagonist, in CMD. Global iNOS deletion prevented CMD along with the associated oxidative stress and diastolic and subclinical systolic dysfunction. The 1400W treatment reversed established CMD and oxidative stress and preserved systolic/diastolic function in mice fed a high-fat, high-sucrose diet. Thus, iNOS may represent a therapeutic target for CMD.

Acute hyperglycemic exacerbation of lung ischemia-reperfusion injury is mediated by receptor for advanced glycation end-products signaling.

The effects of acute hyperglycemia on lung ischemia-reperfusion (IR) injury and the role of receptor for advanced glycation end-products (RAGE) signaling in this process are unknown. The objective of this study was twofold: (1) evaluate the impact of acute hyperglycemia on lung IR injury; and (2) determine if RAGE signaling is a mechanism of hyperglycemia-enhanced IR injury. We hypothesized that acute hyperglycemia worsens lung IR injury through a RAGE signaling mechanism. C57BL/6 wild-type (WT) and RAGE knockout (RAGE (-/-)) mice underwent sham thoracotomy or lung IR (1-h left hilar occlusion and 2-h reperfusion). Acute hyperglycemia was established by dextrose injection 30 minutes before ischemia. Lung injury was assessed by measuring lung function, cytokine expression in bronchoalveolar lavage fluid, leukocyte infiltration, and microvascular permeability via Evans blue dye. Mean blood glucose levels doubled in hyperglycemic mice 30 minutes after dextrose injection. Compared with IR in normoglycemic mice, IR in hyperglycemic mice significantly enhanced lung dysfunction, cytokine expression (TNF-α, keratinocyte chemoattractant, IL-6, monocyte chemotactic protein-1, regulated upon activation, normal T cell expressed and secreted), leukocyte infiltration, and microvascular permeability. Lung injury and dysfunction after IR were attenuated in normoglycemic RAGE (-/-) mice, and hyperglycemia failed to exacerbate IR injury in RAGE (-/-) mice. Thus, this study demonstrates that acute hyperglycemia exacerbates lung IR injury, whereas RAGE deficiency attenuates IR injury and also prevents exacerbation of IR injury in an acute hyperglycemic setting. These results suggest that hyperglycemia-enhanced lung IR injury is mediated, at least in part, by RAGE signaling, and identifies RAGE as a potential, novel therapeutic target to prevent post-transplant lung IR injury.

Displacement-encoded and manganese-enhanced cardiac MRI reveal that nNOS, not eNOS, plays a dominant role in modulating contraction and calcium influx in the mammalian heart.

Within cardiomyocytes, endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) are thought to modulate L-type calcium channel (LTCC) function and sarcoplasmic reticulum calcium cycling, respectively. However, divergent results from mostly invasive prior studies suggest more complex roles. To elucidate the roles of nNOS and eNOS in vivo, we applied noninvasive cardiac MRI to study wild-type (WT), eNOS(-/-), and nNOS(-/-) mice. An in vivo index of LTCC flux (LTCCI) was measured at baseline (Bsl), dobutamine (Dob), and dobutamine + carbacholamine (Dob + CCh) using manganese-enhanced MRI. Displacement-encoded MRI assessed contractile function by measuring circumferential strain (E(cc)) and systolic (dE(cc)/dt) and diastolic (dE(cc)/dt(diastolic)) strain rates at Bsl, Dob, and Dob + CCh. Bsl LTCCI was highest in nNOS(-/-) mice (P < 0.05 vs. WT and eNOS(-/-)) and increased only in WT and eNOS(-/-) mice with Dob (P < 0.05 vs. Bsl). LTCCI decreased significantly from Dob levels with Dob + CCh in all mice. Contractile function, as assessed by E(cc), was similar in all mice at Bsl. With Dob, E(cc) increased significantly in WT and eNOS(-/-) but not nNOS(-/-) mice (P < 0.05 vs. WT and eNOS(-/-)). With Dob + CCh, E(cc) returned to baseline levels in all mice. Systolic blood pressure, measured via tail plethysmography, was highest in eNOS(-/-) mice (P < 0.05 vs. WT and nNOS(-/-)). Mice deficient in nNOS demonstrate increased Bsl LTCC function and an attenuated contractile reserve to Dob, whereas eNOS(-/-) mice demonstrate normal LTCC and contractile function under all conditions. These results suggest that nNOS, not eNOS, plays the dominant role in modulating Ca(2+) cycling in the heart.

Adeno-associated virus serotype 9 administered systemically after reperfusion preferentially targets cardiomyocytes in the infarct border zone with pharmacodynamics suitable for the attenuation of left ventricular remodeling.

BACKGROUND: Adeno-associated virus serotype 9 (AAV9) vectors provide efficient and uniform gene expression to normal myocardium following systemic administration, with kinetics that approach steady-state within 2-3 weeks. However, as a result of the delayed onset of gene expression, AAV vectors have not previously been administered intravenously after reperfusion for post-infarct gene therapy applications. The present study evaluated the therapeutic potential of post-myocardial infarction gene delivery using intravenous AAV9. METHODS: AAV9 vectors expressing firefly luciferase, enhanced green fluorescent protein (eGFP) or extracellular superoxide dismutase genes from the cardiac troponin-T (cTnT) promoter (AcTnTLuc, AcTnTeGFP, AcTnTEcSOD) were employed. AcTnTLuc was administered intravenously at 10 min and at 1, 2 and 3 days post-ischemia/reperfusion (IR), and the kinetics of luciferase expression were assessed with bioluminescence imaging. AcTnTeGFP was used to evaluate the distribution of eGFP expression. High-resolution echocardiography was used to evaluate the effects of AcTnTEcSOD on left ventricular (LV) remodeling when injected 10 min post-IR. RESULTS: Compared to sham animals, luciferase expression at 2 days after vector administration was elevated by four-, 24-, 210- and 213-fold in groups injected at 10 min, 1 day, 2 days and 3 days post-IR, respectively. The expression of cTnT-driven eGFP was strongest in cardiomyocytes bordering the infarct zone. In the efficacy study of EcSOD, post-infarct LV end-systolic and end-diastolic volumes at days 14 and 28 were significantly smaller in the EcSOD group compared to the control. CONCLUSIONS: Systemic administration of AAV9 vectors after IR both elevates and accelerates gene expression that preferentially targets cardiomyocytes in the border zone with pharmacodynamics suitable for the attenuation of LV remodeling.

Monocyte and/or macrophage infiltration of heart after myocardial infarction: MR imaging by using T1-shortening liposomes.

PURPOSE: To test the hypothesis that magnetic resonance (MR) imaging R1 (R1 = 1/T1) mapping after selectively labeling monocytes with a T1-shortening contrast agent in vivo would enable the quantitative measurement of their spatiotemporal kinetics in the setting of infarct healing. MATERIALS AND METHODS: All procedures were performed in mice and were approved by the institutional committee on animal research. One hundred microliters of dual-labeled liposomes (DLLs) containing gadolinium (Gd)-diethylenetriaminepentaacetic acid (DTPA)-bis(stearylamide) and DiI dye were used to label monocytes 2 days before myocardial infarction (MI). MI was induced by occlusion of the left anterior descending coronary artery for 1 hour, followed by reperfusion. MR imaging R1 mapping of mouse hearts was performed at baseline on day -3, on day 0 before MI, and on days 1, 4, and 7 after MI. Mice without labeling were used as controls. ΔR1 was calculated as the difference in R1 between mice with labeling and those without labeling. CD68 immunohistochemistry and DiI fluorescence microscopy were used to confirm that labeled monocytes and/or macrophages infiltrated the postinfarct myocardium. Statistical analysis was performed by using two-way analysis of variance and the unpaired two-sample t test. RESULTS: Infarct zone ΔR1 was slightly but nonsignificantly increased on day 1, maximum on day 4 (P < .05 vs all other days), and started to decrease by day 7 (P < .05 vs days -3, 0, and 1) after MI, closely reflecting the time course of monocyte and/or macrophage infiltration of the infarcted myocardium shown by prior histologic studies. Histologic results confirmed the presence and location of DLL-labeled monocytes and/or macrophages in the infarct zone on day 4 after MI. CONCLUSION: R1 mapping after labeling monocytes with T1-shortening DLLs enables the measurement of post-MI monocyte and/or macrophage spatiotemporal kinetics.

T2 -weighted MRI of post-infarct myocardial edema in mice.

T(2) -weighted, cardiac magnetic resonance imaging (T(2) w CMR) can be used to noninvasively detect and quantify the edematous region that corresponds to the area at risk (AAR) following myocardial infarction (MI). Previously, CMR has been used to examine structure and function in mice, expediting the study of genetic manipulations. To date, CMR has not been applied to imaging of post-MI AAR in mice. We developed a whole-heart, T(2) w CMR sequence to quantify the AAR in mouse models of ischemia and infarction. The ΔB(0) and ΔB(1) environment around the mouse heart at 7 T were measured, and a T(2) -preparation sequence suitable for these conditions was developed. Both in vivo T(2) w and late gadolinium enhanced CMR were performed in mice after 20-min coronary occlusions, resulting in measurements of AAR size of 32.5 ± 3.1 (mean ± SEM)% left ventricular mass, and MI size of 50.1 ± 6.4% AAR size. Excellent interobserver agreement and agreement with histology were also found. This T(2) w imaging method for mice may allow for future investigations of genetic manipulations and novel therapies affecting the AAR and salvaged myocardium following reperfused MI.

A nonpeptide angiotensin II type 2 receptor agonist does not attenuate postmyocardial infarction left ventricular remodeling in mice.

Cardiac overexpression of the angiotensin II type 2 receptor (AT2 R) attenuates left ventricular (LV) remodeling after myocardial infarction (MI) in transgenic mice. We hypothesized that a novel nonpeptide AT2 R agonist, compound 21 (C21), would attenuate post-MI LV remodeling. Fifty-nine mice were studied for 28 days after 1-hour surgical occlusion-reperfusion of the left anterior descending coronary artery. Immediately thereafter, 23 mice received 0.3 mg·kg·d of C21 via Alzet osmotic minipump, 16 received 10 mg·kg·d of the AT1 R antagonist candesartan in drinking water, and 20 were untreated controls. Cardiac magnetic resonance imaging measured ejection fraction (EF), LV end-systolic, and end-diastolic volumes (ESVI and EDVI) indexed to weight serially post MI. Infarct size was measured on day 1 by late gadolinium-enhanced cardiac magnetic resonance imaging. At baseline, heart rate, blood pressure, EDVI, ESVI, and EF were similar between groups. Mean infarct size (42%-45% of LV mass) was similar between groups. C21-treated animals demonstrated adverse LV remodeling (increased EDVI and ESVI at all post-MI time points) compared with control. Candesartan therapy preserved left ventricular EF at day 28 compared with the C21-treated group. Thus, direct stimulation of the AT2 R by C21 at 0.3 mg·kg·d does not attenuate post-MI LV remodeling in reperfused MI in mice.

Contrast-Enhanced Ultrasound Reveals Partial Perfusion Recovery After Hindlimb Ischemia as Opposed to Full Recovery by Laser Doppler Perfusion Imaging.

Mouse models are critical in developing new therapeutic approaches to treat peripheral arterial disease (PAD). Despite decades of research and numerous clinical trials, the efficacy of available therapies is limited. This may suggest shortcomings in our current animal models and/or methods of assessment. We evaluated perfusion measurement methods in a mouse model of PAD by comparing laser Doppler perfusion imaging (LDPI, the most common technique), contrast-enhanced ultrasound (CEUS, an emerging technique) and fluorescent microspheres (conventional standard). Mice undergoing a femoral artery ligation were assessed by LDPI and CEUS at baseline and 1, 4, 7, 14, 28, 60, 90 and 150 d post-surgery to evaluate perfusion recovery in the ischemic hindlimb. Fourteen days after surgery, additional mice were measured with fluorescent microspheres, LDPI, and CEUS. LDPI and CEUS resulted in broadly similar trends of perfusion recovery until 7 d post-surgery. However, by day 14, LDPI indicated full recovery of perfusion, whereas CEUS indicated ∼50% recovery, which failed to improve even after 5 mo. In agreement with the CEUS results, fluorescent microspheres at day 14 post-surgery confirmed that perfusion recovery was incomplete. Histopathology and photoacoustic microscopy provided further evidence of sustained vascular abnormalities.