H11 kinase/heat shock protein 22 deletion impairs both nuclear and mitochondrial functions of STAT3 and accelerates the transition into heart failure on cardiac overload.

BACKGROUND: Cardiac overload, a major cause of heart failure, induces the expression of the heat shock protein H11 kinase/Hsp22 (Hsp22). METHODS AND RESULTS: To determine the specific function of Hsp22 in that context, a knockout mouse model of Hsp22 deletion was generated. Although comparable to wild-type mice in basal conditions, knockout mice exposed to pressure overload developed less hypertrophy and showed ventricular dilation, impaired contractile function, increased myocyte length and accumulation of interstitial collagen, faster transition into heart failure, and increased mortality. Microarrays revealed that hearts from knockout mice failed to transactivate genes regulated by the transcription factor STAT3. Accordingly, nuclear STAT3 tyrosine phosphorylation was decreased in knockout mice. Silencing and overexpression experiments in isolated neonatal rat cardiomyocytes showed that Hsp22 activates STAT3 via production of interleukin-6 by the transcription factor nuclear factor-κB. In addition to its transcriptional function, STAT3 translocates to the mitochondria where it increases oxidative phosphorylation. Both mitochondrial STAT3 translocation and respiration were also significantly decreased in knockout mice. CONCLUSIONS: This study found that Hsp22 represents a previously undescribed activator of both nuclear and mitochondrial functions of STAT3, and its deletion in the context of pressure overload in vivo accelerates the transition into heart failure and increases mortality.

Concurrent vision dysfunctions in convergence insufficiency with traumatic brain injury.

PURPOSE: This study assessed the prevalence of convergence insufficiency (CI) with and without simultaneous vision dysfunctions within the traumatic brain injury (TBI) sample population because although CI is commonly reported with TBI, the prevalence of concurrent visual dysfunctions with CI in TBI is unknown. METHODS: A retrospective analysis of 557 medical records from TBI civilian patients was conducted. Patients were all evaluated by a single optometrist. Visual acuity, oculomotor function, binocular vision function, accommodation, visual fields, ocular health, and vestibular function were assessed. Statistical comparisons between the CI and non-CI, as well as inpatient and outpatient subgroups, were conducted using χ and Z tests. RESULTS: Approximately 9% of the TBI sample had CI without the following simultaneous diagnoses: saccade or pursuit dysfunction; third, fourth, or sixth cranial nerve palsy; visual field deficit; visual spatial inattention/neglect; vestibular dysfunction; or nystagmus. Photophobia with CI was observed in 16.3% (21 of 130), and vestibular dysfunction with CI was observed in 18.5% (24 of 130) of the CI subgroup. Convergence insufficiency and cranial nerve palsies were common and yielded prevalence rates of 23.3% (130 of 557) and 26.9% (150 of 557), respectively, within the TBI sample. Accommodative dysfunction was common within the nonpresbyopic TBI sample, with a prevalence of 24.4% (76 of 314). Visual field deficits or unilateral visual spatial inattention/neglect was observed within 29.6% (80 of 270) of the TBI inpatient subgroup and was significantly more prevalent compared with that of the outpatient subgroup (p < 0.001). Most TBI patients had visual acuities of 20/60 or better in the TBI sample (85%; 473 of 557). CONCLUSIONS: Convergence insufficiency without simultaneous visual or vestibular dysfunctions was observed in about 9% of the visually symptomatic TBI civilian population studied. A thorough visual and vestibular examination is recommended for all TBI patients.

Preemptive conditioning of the swine heart by H11 kinase/Hsp22 provides cardiac protection through inducible nitric oxide synthase.

The second window of ischemic preconditioning (SWOP) provides maximal protection against ischemia through regulation of the inducible nitric oxide synthase (iNOS), yet its application is limited by the inconvenience of the preliminary ischemic stimulus required for prophylaxis. Overexpression of H11 kinase/Hsp22 (Hsp22) in a transgenic mouse model provides cardioprotection against ischemia that is equivalent to that conferred by SWOP. We hypothesized that short-term, prophylactic overexpression of Hsp22 would offer an alternative to SWOP in reducing ischemic damage through a nitric oxide (NO)-dependent mechanism. Adeno-mediated overexpression of Hsp22 was achieved in the area at risk of the left circumflex (Cx) coronary artery in chronically instrumented swine and compared with LacZ controls (n = 5/group). Hsp22-injected myocardium showed an average fourfold increase in Hsp22 protein expression compared with controls and a doubling in iNOS expression (both P < 0.05). Four days after ischemia-reperfusion, regional wall thickening was reduced by 58 ± 2% in the Hsp22 group vs. 82 ± 7% in the LacZ group, and Hsp22 reduced infarct size by 40% (both P < 0.05 vs. LacZ). Treatment with the NOS inhibitor N(G)-nitro-L-arginine (L-NNA) before ischemia suppressed the protection induced by Hsp22. In isolated cardiomyocytes, Hsp22 increased iNOS expression through the transcription factors NF-κB and STAT, the same effectors activated by SWOP, and reduced by 60% H(2)O(2)-mediated apoptosis, which was also abolished by NOS inhibitors. Therefore, short-term, prophylactic conditioning by Hsp22 provides NO-dependent cardioprotection that reproduces the signaling of SWOP, placing Hsp22 as a potential alternative for preemptive treatment of myocardial ischemia.

Inhibition of smooth muscle myosin as a novel therapeutic target for hypertension.

We examined a novel therapeutic approach for hypertension, a small-molecule direct inhibitor of smooth muscle myosin, CK-2018448 (CK-448), which is an N,N'-alkylurea (U.S. Patent Publication 2009-0275537 A1) in conscious dogs with renal hypertension and compared its efficacy with that of a calcium channel blocker, amlodipine. Dogs were instrumented with a miniature left ventricular pressure gauge, an aortic pressure catheter, and ultrasonic flow probes in the ascending aorta and renal and iliac arteries for measurement of cardiac output and regional blood flow. In the hypertensive state, mean arterial pressure increased from 101 ± 3.8 to 142 ± 1.9 mm Hg. At the doses selected, CK-448 and amlodipine increased cardiac output similarly (30 ± 11% versus 33 ± 6.4%) and similarly reduced mean arterial pressure (-22 ± 3.6% versus -16 ± 3.4%) and total peripheral resistance (-36 ± 5.9% versus -37 ± 5.8%). CK-448 had the greatest vasodilator effect in the renal bed, where renal blood flow increased by 46 ± 9.0%, versus 11 ± 3.4% for amlodipine (p < 0.01). CK-488 produced significantly less vasodilation in the limb, where iliac blood flow did not change; in contrast, it rose by 48 ± 12% with amlodipine (p < 0.01). The minimal effects on limb blood flow could limit the development of peripheral edema, an adverse side effect of Ca(2+) channel blockers. In addition, in a rodent model of hypertension, oral administration of a smooth muscle myosin inhibitor resulted in a sustained antihypertensive effect. Thus, the smooth muscle myosin inhibitor's preferential effect on renal blood flow makes this drug mechanism particularly appealing, because many patients with hypertension have renal insufficiency, and patients with heart failure could benefit from afterload reduction coupled with enhanced renal blood flow.

Effects of cardiac overexpression of type 6 adenylyl cyclase affects on the response to chronic pressure overload.

Adenylyl cyclase (AC) type 5 (AC5) and AC type 6 (AC6) are the two major AC isoforms in the heart. Cardiac overexpression of AC6 has been shown to be protective in response to several interventions. In this investigation, we examined the effects of chronic pressure overload in AC6 transgenic (TG) mice. In the absence of any stress, AC6 TG mice exhibited enhanced contractile function compared with their wild-type (WT) littermates, i.e., increased (P < 0.05) left ventricular (LV) ejection fraction (EF) (75 +/- 0.9 vs. 71 +/- 0.5%) and LV dP/dt (7,850 +/- 526 vs. 6,374 +/- 315 mmHg/s). Forskolin (25 microg x kg(-1) x min(-1) for 5 min) increased LVEF more (P < 0.05) in AC6 TG mice (14.8 +/- 1.0%) than in WT mice (7.7 +/- 1.0%). Also, isoproterenol (0.04 microg x kg(-1) x min(-1) for 5 min) increased LVEF more (P < 0.05) in AC6 TG mice (18.0 +/- 1.2%) than in WT mice (11.6 +/- 2.1%). Pressure overload, induced by 4 wk of transverse aortic constriction (TAC), increased the LV weight-to-body weight ratio and myocyte cross-sectional area similarly in both groups, but reduced LVEF more in AC6 TG mice (22%) compared with WT mice (9%), despite the higher starting level of LVEF in AC6 TG mice. LV systolic wall stress increased more in AC6 TG mice than in WT mice, which could be responsible for the reduced LVEF in AC6 TG mice with chronic pressure overload. In addition, LV dP/dt was no longer elevated in AC6 TG mice after TAC compared with WT mice. LV end-diastolic diameter was also greater (P < 0.05) in AC6 TG mice (3.8 +/- 0.07 mm) than in WT mice (3.6 +/- 0.05 mm) after TAC. Thus, in contrast to other interventions previously reported to be salutary with cardiac AC6 overpression, the response to chronic pressure overload was not; actually, AC6 TG mice fared worse than WT mice. The mechanism may be due to the increased LV systolic wall stress in AC6 TG mice with chronic pressure overload.

Cytochrome c oxidase III as a mechanism for apoptosis in heart failure following myocardial infarction.

Cytochrome c oxidase (COX) is composed of 13 subunits, of which COX I, II, and III are encoded by a mitochondrial gene. COX I and II function as the main catalytic components, but the function of COX III is unclear. Because myocardial ischemia affects mitochondrial oxidative metabolism, we hypothesized that COX activity and expression would be affected during postischemic cardiomyopathy. This hypothesis was tested in a monkey model following myocardial infarction (MI) and subsequent pacing-induced heart failure (HF). In this model, COX I protein expression was decreased threefold after MI and fourfold after HF (P < 0.05 vs. sham), whereas COX II expression remained unchanged. COX III protein expression increased 5-fold after MI and further increased 10-fold after HF compared with sham (P < 0.05 vs. sham). The physiological impact of COX III regulation was examined in vitro. Overexpression of COX III in mitochondria of HL-1 cells resulted in an 80% decrease in COX I, 60% decrease in global COX activity, 60% decrease in cell viability, and threefold increase in apoptosis (P < 0.05). Oxidative stress induced by H2O2 significantly (P < 0.05) increased COX III expression. H2O2 decreased cell viability by 47 +/- 3% upon overexpression of COX III, but only by 12 +/- 5% in control conditions (P < 0.05). We conclude that ischemic stress in vivo and oxidative stress in vitro lead to upregulation of COX III, followed by downregulation of COX I expression, impaired COX oxidative activity, and increased apoptosis. Therefore, upregulation of COX III may contribute to the increased susceptibility to apoptosis following MI and subsequent HF.

Strength duration curve for left ventricular epicardial stimulation in patients undergoing cardiac resynchronization therapy.

INTRODUCTION: The strength duration curve has been studied for right ventricular endocardial stimulation. There are differences between left ventricular epicardial and right ventricular endocardial stimulation due to different electrophysiologic properties and different electrode-tissue interface. The strength duration curve for epicardial left ventricular stimulation has not been studied so far. METHODS: One hundred and three patients were studied. The strength duration curves were determined for left ventricular epicardial and right ventricular endocardial stimulation. The studied points were chronaxie, rheobase, and voltage threshold at 0.5 ms. Left ventricular leads Guidant 4512, 4513, 4537, 4538 (unipolar, area 3.5 mm(2); Guidant Corp., St. Paul, MN, USA), Medtronic 4193 (unipolar, area 5.8 mm(2); Medtronic Inc., Minneapolis, MN, USA), Guidant 4518, 4542, 4543 (bipolar, area 4 mm(2)), St. Jude Medical (bipolar, area 4.8 mm(2); St. Jude Medical, St. Paul, MN, USA), and Medtronic 4194 (bipolar, area 5.8 mm(2)) were studied. RESULTS: The Guidant unipolar leads with a distal electrode area of 3.5 mm(2) had a lower chronaxie than the other studied leads. The left ventricular epicardial and right ventricular endocardial chronaxie for 15 patients with Medtronic left ventricular leads 4194 or 4193 (5.8 mm(2)) and right ventricular leads 6947 (5.7 mm(2)) were 0.52 +/- 0.36 ms and 0.62 +/- 0.46 ms (P > 0.05). CONCLUSION: The left ventricular epicardial chronaxie depends on the lead. The left ventricular epicardial chronaxie is similar to the right ventricular endocardial chronaxie for leads with similar electrode stimulation area.

Improvement of cardiac function by a cardiac Myosin activator in conscious dogs with systolic heart failure.

BACKGROUND: Therapy for chronic systolic heart failure (sHF) has improved over the past 2 decades, but the armamentarium of drugs is limited and consequently sHF remains a leading cause of death and disability. In this investigation, we examined the effects of a novel cardiac myosin activator, omecamtiv mecarbil (formerly CK-1827452) in 2 different models of heart failure. METHODS AND RESULTS: Two different models of sHF were used: (1) pacing-induced sHF after myocardial infarction (MI-sHF) and (2) pacing-induced sHF after 1 year of chronic pressure overload left ventricular hypertrophy (LVH-sHF). Omecamtiv mecarbil increased systolic function in sHF dogs, chronically instrumented to measure LV pressure, wall thickness, and cardiac output. Omecamtiv mecarbil, infused for 24 hours, induced a sustained increase without desensitization (P<0.05) in wall thickening (25+/-6.2%), stroke volume (44+/-6.5%) and cardiac output (22+/-2.8%), and decreased heart rate (15+/-3.0%). The major differences between the effect of omecamtiv mecarbil on cardiac function and the effect induced by a catecholamine, for example, dobutamine, is that omecamtiv mecarbil did not increase LV dP/dt but rather increased LV systolic ejection time by 26+/-2.9% in sHF. Another key difference is that myocardial O(2) consumption (MVO(2)), which increases with catecholamines, was not significantly affected by omecamtiv mecarbil. CONCLUSIONS: These results demonstrate that chronic infusion of the cardiac myosin activator, omecamtiv mecarbil, improves LV function in sHF without the limitations of progressive desensitization and increased MVO(2.) This unique profile may provide a new therapeutic approach for patients with sHF.

Proteasome inhibition decreases cardiac remodeling after initiation of pressure overload.

We tested the possibility that proteasome inhibition may reverse preexisting cardiac hypertrophy and improve remodeling upon pressure overload. Mice were submitted to aortic banding and followed up for 3 wk. The proteasome inhibitor epoxomicin (0.5 mg/kg) or the vehicle was injected daily, starting 2 wk after banding. At the end of the third week, vehicle-treated banded animals showed significant (P<0.05) increase in proteasome activity (PA), left ventricle-to-tibial length ratio (LV/TL), myocyte cross-sectional area (MCA), and myocyte apoptosis compared with sham-operated animals and developed signs of heart failure, including increased lung weight-to-TL ratio and decreased ejection fraction. When compared with that group, banded mice treated with epoxomicin showed no increase in PA, a lower LV/TL and MCA, reduced apoptosis, stabilized ejection fraction, and no signs of heart failure. Because overload-mediated cardiac remodeling largely depends on the activation of the proteasome-regulated transcription factor NF-kappaB, we tested whether epoxomicin would prevent this activation. NF-kappaB activity increased significantly upon overload, which was suppressed by epoxomicin. The expression of NF-kappaB-dependent transcripts, encoding collagen types I and III and the matrix metalloprotease-2, increased (P<0.05) after banding, which was abolished by epoxomicin. The accumulation of collagen after overload, as measured by histology, was 75% lower (P<0.05) with epoxomicin compared with vehicle. Myocyte apoptosis increased by fourfold in hearts submitted to aortic banding compared with sham-operated hearts, which was reduced by half upon epoxomicin treatment. Therefore, we propose that proteasome inhibition after the onset of pressure overload rescues ventricular remodeling by stabilizing cardiac function, suppressing further progression of hypertrophy, repressing collagen accumulation, and reducing myocyte apoptosis.

Teaching statistics to clinical research staff in a pharmaceutical company.

Education of clinical research staff in understanding statistical concepts is an area of importance for pharmaceutical companies. This understanding is needed to help them communicate with statisticians using a common language, in designing clinical trials and interpretation of clinical trial results. Such staff has little time for a one-semester or even a one-week continuing education course in statistics. Faced with this reality, we developed a 3-module course,for a total of 1.5 days, which was taught over a period of one month that addresses the needs of this audience. We describe the format and content of the course and provide references that can serve as a resource for teaching such a course.

A bootstrap-based test for establishing noninferiority in clinical trials.

A randomized, active-control clinical trial setting with the objective of testing noninferiority for a continuous response variable is considered. Noninferiority margin is based on the concept of preserving a certain fraction of the active control effect. Noninferiority is established if the ratio of the lower (upper) limit of the two-sided 95% confidence interval for the treatment difference to the estimated mean of the active control is greater (less) than a certain fraction. The nominal significance level is not maintained by the above confidence interval-based noninferiority test. We use bootstrapping to derive an accurate lower (upper) limit of the same confidence interval, which approximates the nominal significance level better and improves the power.