Rajesh Dash

Interactions between phospholamban and β-adrenergic drive may lead to cardiomyopathy and early mortality

Background —Relieving the inhibition of sarcoplasmic reticular function by phospholamban is a major target of &bgr;-adrenergic stimulation. Chronic &bgr;-adrenergic receptor activity has been suggested to be detrimental, on the basis of transgenic overexpression of the receptor or its signaling effectors. However, it is not known whether physiological levels of sympathetic tone, in the absence of preexisting heart failure, are similarly detrimental. Methods and Results —Transgenic mice overexpressing phospholamban at 4-fold normal levels were generated, and at 3 months, they exhibited mildly depressed ventricular contractility without heart failure. As expected, transgenic cardiomyocyte mechanics and calcium kinetics were depressed, but isoproterenol reversed the inhibitory effects of phospholamban on these parameters. In vivo cardiac function was substantially depressed by propranolol administration, suggesting enhanced sympathetic tone. Indeed, plasma norepinephrine levels and the phosphorylation status of phospholamban were elevated, reflecting increased adrenergic drive in transgenic hearts. On aging, the chronic enhancement of adrenergic tone was associated with a desensitization of adenylyl cyclase (which intensified the inhibitory effects of phospholamban), the development of overt heart failure, and a premature mortality. Conclusions —The unique interaction between phospholamban and increased adrenergic drive, elucidated herein, provides the first evidence that compensatory increases in catecholamine stimulation can, even in the absence of preexisting heart failure, be a primary causative factor in the development of cardiomyopathy and early mortality.

Differential regulation of p38 mitogen-activated protein kinase mediates gender-dependent catecholamine-induced hypertrophy

OBJECTIVE Exogenous catecholamine exposure has been associated with p38 mitogen-activated protein kinase (MAPK) and cardiac hypertrophy. In this study, we investigated the regulation of p38 MAPK in cardiac remodeling elicited by endogenous adrenergic mechanisms. METHODS Transgenic male and female mice with fourfold phospholamban (PLB) overexpression exhibited enhanced circulating norepinephrine (NE), as a physiological compensatory mechanism to attenuate PLBs inhibitory effects. This enhanced noradrenergic state resulted in left ventricular hypertrophy/dilatation and depressed function. RESULTS Male transgenics exhibited ventricular hypertrophy and mortality at 15 months, concurrent with cardiac p38 MAPK activation. Female transgenics, despite similar contractile dysfunction, displayed a temporal delay in p38 activation, hypertrophy, and mortality (22 months), which was associated with sustained cardiac levels of MAP Kinase Phosphatase-1 (MKP-1), a potent inhibitor of p38. At 22 months, decreases in cardiac MKP-1 were accompanied by increased levels of p38 activation. In vitro studies indicated that preincubation with 17-beta-estradiol induced high MKP-1 levels, which precluded NE-induced p38 activation. CONCLUSION These findings suggest that norepinephrine-induced hypertrophy is linked closely with p38 MAP kinase activation, which can be endogenously modulated through estrogen-responsive regulation of MKP-1 expression.

Astressin, a novel and potent CRF antagonist, is neuroprotective in the hippocampus when administered after a seizure

Corticotropin-releasing factor (CRF), the principle hypothalamic regulator of the adrenocortical axis, also functions as a neurotransmitter. In this latter role, CRF causes electrophysiological activation and epileptiform activity in various brain regions. That finding, coupled with the observation that CRF mRNA is induced in endangered brain regions following necrotic insults, suggests that the peptide might contribute to necrotic neuron loss. Supporting that, a number of studies have shown that CRF antagonists decrease ischemic or excitotoxic damage to neurons. In the present report, we demonstrate the considerable neuroprotective potential of a novel and potent CRF antagonist, astressin, against kainic acid-induced excitotoxic seizures. Intracerebroventricular infusion of the peptide both 30 min before and 10 min after seizures decreased damage in some hippocampal cell fields by as much as 84%, a magnitude of protection greater than reported for other CRF antagonists against other models of necrotic neuronal injury. Administration of astressin was done against both local microinfusion (0.035 microgram) or systemic infusion (10 mg/kg body weight) of the excitotoxin; furthermore, the peptide protected even if administered only 10 min following excitotoxin exposure. This fulfills a critical prerequisite for any eventual therapeutic use of CRF antagonists, namely that they need not be administered in anticipation of a neurological insult.

Herpes simplex virus vector system: analysis of its in vivo and in vitro cytopathic effects

With its natural propensity to infect and establish life-long latency in neurons, herpes simplex virus type 1 (HSV-1) has been successfully employed by various laboratories as vectors for gene transfer into neurons. However, analysis of its cytopathic effects in vivo and in vitro has been limited. In this study, we examined the cytopathic effects of 2 HSV-1 alpha 4 mutants (ts756 and d120) on adult rat hippocampus and striatum and of d120 on hippocampal neurons in culture. We assessed damage by stringent counting of surviving neurons after infection and demonstrated that while neither ts756 nor d120 infection resulted in any gross anatomical or behavioral changes of the animals, ts756, but not d120, produced a significant amount of damage in the CA4 cell field and dentate gyrus of the hippocampus. Thus, since crude examination is insufficient to detect subtle but significant degrees of neuron loss, the cytopathic effects of HSV or any vector system must be carefully analyzed. Furthermore, we also observed that uninfected cell lysates damaged neurons, both in vivo and in vitro. This cytotoxicity occurred within the first 24 h post-inoculation and probably arose through the activation of glutamate receptors. For the preparation of HSV vectors, purification of the virus from soluble cellular components by a simple pelleting step can significantly decrease such acute toxicity.

A molecular MRI probe to detect treatment of cardiac apoptosis in vivo.

Cell death by apoptosis is critical in myocardial diseases, and noninvasive detection of early, reversible apoptosis might be useful clinically. Exogenous Annexin‐V (ANX) protein binds membrane phosphatidylserine, which is externalized in early apoptosis. A molecular MRI probe was constructed with superparamagnetic iron oxide (SPIO) conjugated to recombinant human ANX (ANX‐SPIO). Apoptosis was induced with doxorubicin, a cardiotoxic cancer drug, in culture in neonatal rat ventricular myocytes, cardiac fibroblasts, and mesenchymal stem cells, and in vivo in the mouse heart. ANX‐SPIO was validated using T2*‐weighted 3T MRI. ANX‐SPIO produced T2* signal loss, reflecting iron content, that correlated highly with independent apoptosis markers; bound with high affinity to apoptotic myocytes by competition assay (Ki 69 nM); detected apoptosis in culture much earlier than did TUNEL stain; and revealed fibroblast resistance to apoptosis. With apoptosis in vivo, ANX‐SPIO produced diffuse myocardial T2* signal loss that correlated with increased iron stain and caspase activity. Treatment with an alpha‐1‐adrenergic agonist in vivo reversed apoptosis and eliminated the ANX‐SPIO MRI signal. It is concluded that cardiac MRI of ANX‐SPIO detects early, nonischemic cardiac apoptosis in culture and in vivo, and can identify reversibly injured cardiac cells in diseased hearts, when treatment is still possible. Magn Reson Med, 2011.

Low-Dose FK506 (Tacrolimus) in End-Stage Pulmonary Arterial Hypertension

To the Editor: Despite recent advances in therapy, pulmonary arterial hypertension (PAH), characterized by occlusive vasculopathy of the pulmonary arteries, remains a progressive disease without a cure (1–3). Although currently approved PAH medications have not demonstrated antiremodeling properties in humans, novel antiproliferative strategies have shown some benefits and also raised safety concerns (4–6); for example, none target a genetic predisposition of PAH: dysfunctional bone morphogenetic protein receptor 2 (BMPR2) signaling. Loss-of-function mutations in BMPR2 in patients with familial and idiopathic PAH (7–9) are associated with increased pulmonary vasculopathy (10). Furthermore, reduced BMPR2 expression is observed even in patients without a mutation, reinforcing the importance of decreased BMPR2 in PAH (11). In a high-throughput screen of 3,600 drugs approved by the U.S. Food and Drug Administration, we identified low-dose FK506 (tacrolimus) as a potent BMPR2 activator that reversed experimental PAH (12). We therefore hypothesized that low-dose FK506 would be beneficial in patients with PAH by increasing BMPR2 signaling. On the basis of these findings, we initiated a randomized, double-blind, placebo-controlled phase IIa trial (FK506 [Tacrolimus] in Pulmonary Arterial Hypertension [TransformPAH]) to evaluate the safety and tolerability of FK506 in stable patients with PAH. Here, we report our clinical experience with compassionate use of low-dose FK506 in three patients with end-stage PAH who did not qualify for our trial because of the severity of their illness (patient details are provided in the online supplement). We assessed traditional clinical parameters, New York Heart Association (NYHA) functional class, 6-minute-walk distance, serologic biomarkers, and hospital admissions, as well as protocolized cardiac magnetic resonance imaging assessed by blinded readers (13, 14). All patients continued receiving stable doses of PAH medication and diuretics throughout the 12-month period. The TransformPAH clinical trial was registered with www.clinicaltrials.gov (NCT 01647945). Patient 1 Patient 1 is a 36-year-old historically athletic woman, NYHA-IV, with rapidly progressive idiopathic PAH requiring rapid up-titration of epoprostenol and the addition of sildenafil and ambrisentan for recurrent hospitalizations for right ventricular (RV) failure (Figure 1). Despite aggressive treatment, she still reported NYHA-III/IV symptoms, an elevated N-terminal-pro-B type natriuretic peptide, and a Registry to Evaluate Early and Long-Term PAH Disease Management (REVEAL) risk score of 11, stratifying her as high risk with a potential 1-year mortality risk of 15–30% (3, 15). She was listed for lung transplantation. At that time, she was offered compassionate treatment with FK506 (goal trough blood level, 1.5–2.5 ng/ml). Figure 1. Timeline of symptoms, clinical parameters, events, and therapies for patients 1–3 before and after initiation of FK506. Registry to Evaluate Early and Long-Term PAH Disease Management (REVEAL) risk score, %1-year survival: score 1–7 = 95–100% ... Within 1 month of FK506 initiation, she reported substantial improvement in symptoms (Figure 2). Within 2 months, she was placed on hold for transplantation by the lung transplant team. After 3 months, her 6-minute-walk distance improved by 100 m, her N-terminal-pro-B type natriuretic peptide decreased more than 50%, and she reported NYHA-I symptoms (Figure 1; see Figure E1A in the online supplement). During the 12-month period, cardiac magnetic resonance imaging showed a stable RV ejection fraction, RV end-diastolic volume index, and cardiac index (Figure E1B). Her REVEAL risk score decreased to 3 (range, 3–6), placing her in the low-risk category (Figure 1). Although the 12 months before FK506 were characterized by three hospitalizations for RV failure, the subsequent 12 months were free of any PAH-associated hospitalizations (Figure 1). At the time of this submission, the patient is 27 months from the initiation of FK506, continues to report NYHA-II symptoms, and has been free from hospitalization or clinical deterioration. Figure 2. Biomarkers for patients 1–3 before and after initiation of FK506. (A) Bone morphogenetic protein receptor 2 (BMPR2) messenger RNA (mRNA)/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in peripheral blood mononuclear cells (PBMCs) of healthy ... Patient 2 Patient 2 is a 50-year-old woman with end-stage systemic sclerosis–associated PAH receiving intravenous treprostinil, sildenafil, and ambrisentan, as well as an intravenous dopamine infusion for end-stage RV failure and hypotension. The patient continued to report NYHA-III/IV symptoms and had an elevated N-terminal-pro-B type natriuretic peptide (range, 4,926–15,161 pg/ml) and four hospitalizations for progressive RV failure and palliative paracenteses (Figure 1). Given the lack of further therapeutic options, she was offered FK506. Cardiac magnetic resonance imaging at baseline and 3 and 6 months showed substantial improvement in RV ejection fraction, stable RV end-diastolic volume index, and improvement in right ventricular stroke volume index and cardiac index (Figure E1B), with a reduction back to baseline at 12 months. Her REVEAL risk score decreased from 12 to 11. At 12-month follow-up, she had stable NYHA-III symptoms, a 94-m increase in 6-minute-walk distance, 30% reduction in her N-terminal-pro-B type natriuretic peptide, and no PAH-related hospitalizations since being on FK506 (Figure 1). The patient is currently 26 months post-FK506 initiation and has not experienced further clinical deterioration. Patient 3 Patient 3 is a 55-year-old woman with severe end-stage drugs-and-toxins–associated PAH, NYHA-III/IV, who is receiving high-dose intravenous treprostinil and sildenafil, has an intolerance to endothelin receptor antagonists, is listed for lung transplantation, and was offered FK506. Despite initial symptomatic improvement (Figure 1), the patient voluntarily discontinued FK506 after 4.5 months. Unfortunately, during the ensuing 7 months, she showed progressive clinical worsening, culminating in an intensive care unit admission for RV failure and large pericardial effusion. On the patient’s wish, she was restarted on FK506 and is currently 12 months after her second FK506 initiation, feeling much better, with compensated NYHA-II symptoms and without any further hospital admission for RV failure. Serologic Biomarkers None of the three patients had mutations in BMPR2, SMAD9, or caveolin-1. We measured BMPR2 expression and specific BMPR2-associated genes and molecules (Id1 [inhibitor of differentiation 1], Smurf-1 [SMAD-specific E3 ubiquitin protein ligase 1], IL-6, LIMK1 [LIM domain kinase 1], Cofilin-1, and microRNAs 21 and 27a) at baseline and 3, 6, and 12 months into FK506 treatment in patients versus healthy controls (n = 12) (see online supplement). Patients had significantly lower BMPR2 messenger RNA expression at baseline (Figure 2), with near normalization of BMPR2 and associated genes after 12 months of FK506 treatment. Strikingly, patient 3, who stopped FK506 after 4.5 months and who worsened clinically during the following 7 months, showed a 12-month BMPR2 profile that was opposite that of patients still receiving FK506 therapy. Discussion Our results suggest a potential clinical benefit of low-dose FK506 in end-stage PAH, judged by the patients’ marked clinical response, stabilization in cardiac function, and freedom from hospitalization for RV failure. Despite the overall positive experience, we caution that these findings are highly preliminary. The efficacy of this therapy must be validated in appropriate, well-designed, prospective clinical trials. Our choice of low-dose FK506 was based on data from preclinical studies (12) and the desire to avoid major immunosuppressive adverse effects in patients with indwelling lines. We did not observe an increase in line sepsis or opportunistic infections. We also did not observe serious adverse effects of posterior reversible encephalopathy syndrome, acute kidney injury or worsening of creatinine, an elevated systemic blood pressure, hyperglycemia, hyperkalemia, anemia, or a change in white blood cell count. The currently underway clinical phase IIa trial will address safety and tolerability in greater detail, as even low-dose immunosuppression over time can lead to complications. This is the first study in patients with PAH that repurposes FK506 to increase BMPR2 signaling. The changes in serologic biomarkers are encouraging and show that we have indeed targeted BMPR2 in patients with reduced levels of BMPR2. It will be of interest to determine whether the same effect can be achieved in patients with documented mutations and whether a subset of patients is particularly sensitive to the beneficial effects of this strategy and could therefore be identified up front as potential “responders” on the basis of BMPR2 levels.

Near Infrared Imaging and Photothermal Ablation of Vascular Inflammation Using Single-Walled Carbon Nanotubes

Background Macrophages are critical contributors to atherosclerosis. Single-walled carbon nanotubes (SWNTs) show promising properties for cellular imaging and thermal therapy, which may have application to vascular macrophages. Methods and Results In vitro uptake and photothermal destruction of mouse macrophage cells (RAW264.7) were performed with SWNTs (14.7 nmol/L) exposed to an 808-nm light source. SWNTs were taken up by 94±6% of macrophages, and light exposure induced 93±3% cell death. In vivo vascular macrophage uptake and ablation were then investigated in carotid-ligated FVB mice (n=33) after induction of hyperlipidemia and diabetes. Two weeks postligation, near-infrared fluorescence (NIRF) carotid imaging (n=12) was performed with SWNT-Cy5.5 (8 nmol of Cy5.5) given via the tail vein. Photothermal heating and macrophage apoptosis were evaluated on freshly excised carotid arteries (n=21). NIRF of SWNTs showed higher signal intensity in ligated carotids compared with sham, confirmed by both in situ and ex vivo NIRF imaging (P<0.05, ligation versus sham). Immunofluorescence staining showed colocalization of SWNT-Cy5.5 and macrophages in atherosclerotic lesions. Light (808 nm) exposure of freshly excised carotids showed heating and induction of macrophage apoptosis in ligated left carotid arteries with SWNTs, but not in control groups without SWNTs or without light exposure. Conclusions Carbon nanotubes accumulate in atherosclerotic macrophages in vivo and provide a multifunctional platform for imaging and photothermal therapy of vascular inflammation.

A Herpes Simplex Virus Vector Overexpressing the Glucose Transporter Gene Protects the Rat Dentate Gyrus from an Antimetabolite Toxin

The use of herpes simplex virus vectors offers an attractive means for the in vitro and in vivo transfer of novel genes into postmitotic neurons. Such an approach allows for the introduction of genes with the potential to protect neurons from necrotic insults. Toward that end, we have previously constructed a bicistronic herpes viral vector expressing the gene for the Glut-1 rat brain glucose transporter (GT), along with the Escherichia coli lacZ reporter gene. We observed that this vector enhances glucose uptake both in primary hippocampal cultures and in the hippocampus itself. Moreover, we have found that this vector will protect a variety of types of cultured neurons from necrotic insults and protect hippocampal neurons in vivo from seizure-induced damage. In the present report, we further demonstrate the neuroprotective potential of this GT-expressing vector. 3-Acetylpyridine, an electron transport uncoupler which is preferentially toxic to the dentate gyrus, was microinfused into the dorsal hippocampus of rats. Infection of dentate neurons with GT vectors at the time of exposure to the toxin significantly decreased damage, whereas infection with a physiologically neutral control vector did not. Moreover, there was a window of opportunity for this intervention, as microinfusion of the GT-expressing vector up to 1 h, but not 4 h, after the insult was still neuroprotective.

A Novel Stress Echocardiography Pattern for Myocardial Bridge With Invasive Structural and Hemodynamic Correlation

Background Patients with a myocardial bridge (MB) and no significant obstructive coronary artery disease (CAD) may experience angina presumably from ischemia, but noninvasive assessment has been limited and the underlying mechanism poorly understood. This study seeks to correlate a novel exercise echocardiography (EE) finding for MBs with invasive structural and hemodynamic measurements. Methods and Results Eighteen patients with angina and an EE pattern of focal end‐systolic to early‐diastolic buckling in the septum with apical sparing were prospectively enrolled for invasive assessment. This included coronary angiography, left anterior descending artery (LAD) intravascular ultrasound (IVUS), and intracoronary pressure and Doppler measurements at rest and during dobutamine stress. All patients were found to have an LAD MB on IVUS. The ratios of diastolic intracoronary pressure divided by aortic pressure at rest (Pd/Pa) and during dobutamine stress (diastolic fractional flow reserve [dFFR]) and peak Doppler flow velocity recordings at rest and with stress were successfully performed in 14 patients. All had abnormal dFFR (≤0.75) at stress within the bridge, distally or in both positions, and on average showed a more than doubling in peak Doppler flow velocity inside the MB at stress. Seventy‐five percent of patients had normalization of dFFR distal to the MB, with partial pressure recovery and a decrease in peak Doppler flow velocity. Conclusions A distinctive septal wall motion abnormality with apical sparing on EE is associated with a documented MB by IVUS and a decreased dFFR. We posit that the septal wall motion abnormality on EE is due to dynamic ischemia local to the compressed segment of the LAD from the increase in velocity and decrease in perfusion pressure, consistent with the Venturi effect.

In vivo molecular MRI of cell survival and teratoma formation following embryonic stem cell transplantation into the injured murine myocardium

Embryonic stem cells (ESCs) have shown the potential to restore cardiac function after myocardial injury. Superparamagnetic iron oxide nanoparticles (SPIO) have been widely employed to label ESCs for cellular MRI. However, nonspecific intracellular accumulation of SPIO limits long‐term in vivo assessment of the transplanted cells. To overcome this limitation, a novel reporter gene (RG) has been developed to express antigens on the ESC surface. By employing SPIO‐conjugated monoclonal antibody against these antigens (SPIO‐MAb), the viability of transplanted ESCs can be detected in vivo. This study aims to develop a new molecular MRI method to assess in vivo ESC viability, proliferation, and teratoma formation. The RG is designed to express 2 antigens (hemagglutinin A and myc) and luciferase on the ESC surface. The two antigens serve as the molecular targets for SPIO‐MAb. The human and mouse ESCs were transduced with the RG (ESC‐RGs) and transplanted into the peri‐infarct area using the murine myocardial injury model. In vivo MRI was performed following serial intravenous administration of SPIO‐MAb. Significant hypointense signal was generated from the viable and proliferating ESCs and subsequent teratoma. This novel molecular MRI technique enabled in vivo detection of early ESC‐derived teratoma formation in the injured murine myocardium. Magn Reson Med, 2011.