Charles R. Bridges

Cardiac Surgery in Jehovah's Witness Patients: Ten-Year Experience

BACKGROUNDnCardiac surgery in Jehovahs Witnesses poses unique challenges. We have developed a comprehensive multimodality program for these patients and have obtained excellent results.nnnMETHODSnNinety-one Jehovahs Witness patients underwent cardiac surgery between 2000 and 2010. Preoperative, intraoperative, and postoperative considerations in the conduct of bloodless surgery in the Jehovahs Witness population are discussed. Mortality for isolated coronary artery bypass graft surgery and isolated aortic valve replacement was compared with predicted mortality from The Society of Thoracic Surgeons (STS) risk models. Perioperative outcomes were stratified by urgent and elective status of operations.nnnRESULTSnMean age was 65±12.4 years. Comorbid conditions included hypertension (84.6%), diabetes mellitus (48.4%), previous myocardial infarction (23.1%), chronic lung disease (38.5%), peripheral vascular disease (20.9%), and renal failure (11%). In-hospital mortality was 5.5% (n=5). Mortality for isolated coronary artery bypass graft surgery and isolated aortic valve replacement was 2.2% (observed to expected ratio=1.05, 95% confidence interval: 0 to 3.02) and 5.6% (observed to expected=1.46, 95% confidence interval: 0 to 3.76), respectively. Other complications included reoperation (all=8.8%, cardiac=2.2%), sepsis (2.2%), sternal wound infection (1.1%), transient ischemic attack (1.1%), renal failure requiring dialysis (1.1%), and prolonged ventilation (18.7%). Major complication rates were not significantly different between the elective group and the urgent group.nnnCONCLUSIONSnBloodless cardiac surgery in Jehovahs Witness patients can be performed with excellent outcomes in both elective and urgent situations. Mortality rates for isolated coronary artery bypass graft surgery and isolated aortic valve replacement are within the expected 95% confidence intervals of STS predicted mortality.

Safety and efficacy of high-dose adeno-associated virus 9 encoding sarcoplasmic reticulum Ca2+ adenosine triphosphatase delivered by molecular cardiac surgery with recirculating delivery in ovine ischemic cardiomyopathy

OBJECTIVEnTherapeutic safety and efficacy are the basic prerequisites for clinical gene therapy. We investigated the effect of high-dose molecular cardiac surgery with recirculating delivery (MCARD)-mediated adeno-associated virus 9 (AAV9)/sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA2a) gene delivery on clinical parameters, oxidative stress, humoral and cellular immune responses, and cardiac remodeling.nnnMETHODSnIschemic cardiomyopathy was generated in a sheep model. The sheep were assigned to 1 of 2 groups: control (n = 10) and study (MCARD, n = 6). The control group underwent no intervention and the study group received 10(14) genome copies of AAV9/SERCA2a 4 weeks after infarction.nnnRESULTSnOur ischemic model produced reliable infarcts leading to heart failure. The baseline ejection fraction in the MCARD group was 57.6% ± 1.6% versus 61.2% ± 1.9% in the control group (P > .05). At 12 weeks after infarction, the MCARD group had superior left ventricular function compared with the control group: stroke volume index, 46.6 ± 1.8 versus 35.8 ± 2.5 mL/m(2) (P < .05); ejection fraction, 46.2% ± 1.9% versus 38.7% ± 2.5% (P < .05); and left ventricular end-systolic and end-diastolic dimensions, 41.3 ± 1.7 versus 48.2 ± 1.4 mm and 51.2 ± 1.5 versus 57.6 ± 1.7 mm, respectively (P < .05). The markers of oxidative stress were significantly reduced in the infarct zone in the MCARD group. No positive T-cell-mediated immune response was seen in the MCARD group at any point. Myocyte hypertrophy was also significantly attenuated in the MCARD group compared with the control group.nnnCONCLUSIONSnCardiac overexpression of the SERCA2a gene by way of MCARD is a safe therapeutic intervention. It significantly improves left ventricular function, decreases markers of oxidative stress, abrogates myocyte hypertrophy, arrests remodeling, and does not induce a T-cell-mediated immune response.

The role of microRNAs in cardiac development and regenerative capacity

The mammalian heart has long been considered to be a postmitotic organ. It was thought that, in the postnatal period, the heart underwent a transition from hyperplasic growth (more cells) to hypertrophic growth (larger cells) due to the conversion of cardiomyocytes from a proliferative state to one of terminal differentiation. This hypothesis was gradually disproven, as data were published showing that the myocardium is a more dynamic tissue in which cardiomyocyte karyokinesis and cytokinesis produce new cells, leading to the hyperplasic regeneration of some of the muscle mass lost in various pathological processes. microRNAs have been shown to be critical regulators of cardiomyocyte differentiation and proliferation and may offer the novel opportunity of regenerative hyperplasic therapy. Here we summarize the relevant processes and recent progress regarding the functions of specific microRNAs in cardiac development and regeneration.

Cardiac Surgical Delivery of the Sarcoplasmic Reticulum Calcium ATPase Rescues Myocytes in Ischemic Heart Failure

BACKGROUNDnThe sarcoplasmic reticulum calcium ATPase (SERCA2a) is an important molecular regulator of contractile dysfunction in heart failure. Gene transfer of SERCA2a mediated by molecular cardiac surgery with recirculating delivery (MCARD) is a novel and clinically translatable strategy.nnnMETHODSnIschemic heart failure was induced by ligation of OM1 and OM2 in 14 sheep. Seven sheep underwent MCARD-mediated AAV1-SERCA2a delivery 4 weeks after myocardial infarction, and seven sheep served as untreated controls. Magnetic resonance imaging-based mechanoenergetic studies were performed at baseline, 3 weeks, and 12 weeks after infarction. Myocyte apoptosis was quantified by Tdt-mediated nick-end labeling assay. Myocyte cross-sectional area and caspase-8 and caspase-9 activity was measured with imaging software, specific fluorogenic peptides, and immunohistochemistry.nnnRESULTSnMCARD-mediated AAV1-SERCA2a gene delivery resulted in robust cardiac-specific SERCA2a expression and stable improvements in global and regional contractility. There were significantly higher stroke volume index, left ventricular fractional thickening, and ejection fraction at 12 weeks in the MCARD group than in the control group (30 ± 3 vs 21 ± 2 mL/m(2); 12% ± 5% vs 3% ± 3%; and 43 ± 4 vs 32 ± 4, respectively, all p < 0.05). Apoptotic myocytes were observed more frequently in the control group than in the MCARD-SERCA2a group (0.57.2 ± 0.16 AU vs 0.32.4 ± 0.08 AU, p < 0.05). MCARD-SERCA2a also resulted in decreased caspase-8 and caspase-9 expression and decreased myocyte area in the border zone of transgenic sheep compared with control sheep (14.6% ± 1.2% vs 2.9% ± 0.7%; 18.2% ± 1.9% vs 8.6% ± 1.4%; and 102.1 ± 3.8 μm(2) vs 88.1 ± 3.6 μm(2), all p < 0.05).nnnCONCLUSIONSnMCARD-mediated SERCA2a delivery results in robust cardiac specific gene expression, improved contractility, and a decrease in both myocyte apoptosis and myocyte hypertrophy.

A Needleless Liquid Jet Injection Delivery Method for Cardiac Gene Therapy: a Comparative Evaluation Versus Standard Routes of Delivery Reveals Enhanced Therapeutic Retention and Cardiac Specific Gene Expression

This study evaluates needleless liquid jet method and compares it with three common experimental methods: (1) intramuscular injection (IM), (2) left ventricular intracavitary infusion (LVIC), and (3) LV intracavitary infusion with aortic and pulmonary occlusion (LVIC-OCCL). Two protocols were executed. First (nu2009=u200924 rats), retention of dye was evaluated 10xa0min after delivery in an acute model. The acute study revealed the following: significantly higher dye retention (expressed as % myocardial cross-section area) in the left ventricle in both the liquid jet [52u2009±u20094] % and LVIC-OCCL [58u2009±u20093] % groups pu2009<u20090.05 compared with IM [31u2009±u20098] % and LVIC [35u2009±u20094] %. In the second (nu2009=u200916 rats), each animal received adeno-associated virus encoding green fluorescent protein (AAV.EGFP) at a single dose with terminal 6-week endpoint. In the second phase with AAV.EGFP at 6xa0weeks post-delivery, a similar trend was found with liquid jet [54u2009±u20095] % and LVIC-OCCL [60u2009±u20098] % featuring more LV expression as compared with IM [30u2009±u20099] % and LVIC [23u2009±u20099] %. The IM and LVIC-OCCL cross sections revealed myocardial fibrosis. With more detailed development in future model studies, needleless liquid jet delivery offers a promising strategy to improve direct myocardial delivery.

Anti-inflammatory loaded poly-lactic glycolic acid nanoparticle formulations to enhance myocardial gene transfer: an in-vitro assessment of a drug/gene combination therapeutic approach for direct injection

BackgroundCardiac gene therapy for heart disease is a major translational research area with potential, yet problems with safe and efficient gene transfer into cardiac muscle remain unresolved. Existing methodology to increase vector uptake include modifying the viral vector, non-viral particle encapsulation and or delivery with device systems. These advanced methods have made improvements, however fail to address the key problem of inflammation in the myocardium, which is known to reduce vector uptake and contribute to immunogenic adverse events. Here we propose an alternative method to co-deliver anti-inflammatory drugs in a controlled release polymer with gene product to improve therapeutic effects.MethodsA robust, double emulsion production process was developed to encapsulate drugs into nanoparticles. Briefly in this proof of concept study, aspirin and prednisolone anti-inflammatory drugs were encapsulated in various poly-lactic glycolic acid polymer (PLGA) formulations. The resultant particle systems were characterized, co-delivered with GFP plasmid, and evaluated in harvested myocytes in culture for uptake.ResultsHigh quality nanoparticles were harvested from multiple production runs, with an average 64u2009±u200910xa0mg yield. Four distinct particle drug system combinations were characterized and evaluated in vitro: PLGA(50:50) Aspirin, PLGA(65:35) Prednisolone, PLGA(65:35) Aspirin and PLGA(50:50) Prednisolone Particles consisted of spherical shape with a narrow size distribution 265u2009±u2009104xa0nm as found in scanning electron microscopy imaging. Prednisolone particles regardless of PLGA type were found on averageu2009≈u2009100xa0nm smaller than the aspirin types. All four groups demonstrated high zeta potential stability and re-constitution testing prior to in vitro. In vitro results demonstrated co uptake of GFP plasmid (green) and drug loaded particles (red) in culture with no incidence of toxicity.ConclusionsNano formulated anti-inflammatories in combination with standalone gene product therapy may offer a clinical solution to maximize cardiac gene therapy product effects while minimizing the risk of the host response in the inflammatory myocardial environment.

MCARD-Mediated Gene Transfer of GRK2 Inhibitor in Ovine Model of Acute Myocardial Infarction

Abstractβ-Adrenergic receptor (βAR) dysfunction in acute myocardial infarction (MI) is associated with elevated levels of the G-protein-coupled receptor kinase-2 (GRK2), which plays a key role in heart failure progression. Inhibition of GRK2 via expression of a peptide βARKct transferred by molecular cardiac surgery with recirculating delivery (MCARD) may be a promising intervention. Five sheep underwent scAAV6-mediated MCARD delivery of βARKct, and five received no treatment (control). After a 3-week period, the branch of the circumflex artery (OM1) was ligated. Quantitative PCR data showed intense βARKct expression in the left ventricle (LV). Circumferential fractional shortening was 23.4u2009±u20097.1xa0% (baseline) vs. −2.9u2009±u20095.2xa0% (pu2009<u20090.05) in the control at 10xa0weeks. In the MCARD-βARKct group, this parameter was close to baseline. The same trend was observed with LV wall thickening. Cardiac index fully recovered in the MCARD-βARKct group. LV end-diastolic volume and LV end-diastolic pressure did not differ in both groups. MCARD-mediated βARKct gene expression results in preservation of regional and global systolic function after acute MI without arresting progressive ventricular remodeling.

Letter to the editor: Characterizing preclinical model of ischemic heart failure: difference between LAD and LCx infarctions

to the editor: We read with great interest the recently published article of Ishikawa and colleagues ([1][1]). The authors established heart failure (HF) models via left anterior descending coronary artery (LAD) and left circumflex artery (LCx) occlusion. We would like to address several issues that

Swallow syncope after cardiac surgery in a sheep.

To the Editor: We report swallow syncope, a relatively rare, vagally mediated syndrome that occurred in an otherwise healthy 1-year old, 36 kg Dorset sheep, after experimental cardiac surgery with use of cardiopulmonary bypass for cardiac isolation for gene delivery. The gene vector was washed out immediately after bypass and did not enter the systemic circulation, which was shown with real time polymerase chain reaction analysis of peripheral blood and tissue samples. General anesthesia lasted 6.5 hours and included ketamine (4 mg/kg), diazepam (0.5 mg/kg), propofol (6 mg/kg), fentanyl infusion (0.1 µg/kg/min), preoperative fentanyl patch (50 µg/h), and 2–3% isoflurane in oxygen. Intravenous fluids (1.33 mL/min; Normosol-M and 5% Dextrose), oxygen (5 L/min) by nasal cannula until the sheep was able to maintain a pulse oximetry value ≥95%, and antibiotics (potassium penicillin 22,000 U/kg IV) were administered postoperatively. In the first 14 hours after extubation, the sheep was continuously monitored with ECG, noninvasive blood pressure, pulse oximetry, temperature, central venous pressure, respiratory rate, and every 2 hours arterial blood gases. Eight hours after an uneventful recovery from general anesthesia 2 syncopal episodes occurred after eating pellets and spontaneous recovery subsequently occurred within 3–5 minutes. Syncope was characterized by transient loss of consciousness and postural tone with temporary loss of vision and hearing. Syncope was not accompanied by severe bradycardia (sick sinus syndrome), or seizures with heart block or another type of arrhythmia (Adams–Stokes syndrome), or disturbance of acid–base and electrolyte balance, and was not induced by medications. Rectal temperature and respiratory rate were stable and no arrhythmias were detected by ECG. n nGiven that this sheep was administered a large dose of fentanyl during the surgical procedure, after the 2nd syncopal episode, naloxone (0.01 mg/kg subcutaneously) was administered to reverse possible narcotic depression and consequences of anesthesia. The next day, episodic feeding induced ataxia and inability to stand were observed and continued once a day for 14 days. These events occurred during attempts to swallow large boluses of hay and >100 g pellets. Chewing and swallowing small amounts of hay and pellets produced no syncopatic response. Recovery after the 3rd day typically occurred within 10–20 seconds with resumption of seemingly normal behavior and continued interest in eating. During the first 2 episodes, noninvasive blood pressure was slightly increased (125/94 and 135/88 mmHg) and was accompanied by moderate tachycardia (mean, 136 beats/min) and then mild bradycardia (mean, 86 beats/min), but was without evidence of arrhythmia or AV block. Scheduled feedings supervised by a veterinary technician every 6 hours with small amounts of alfalfa hay and pellets were continued as tolerated with less pronounced syncopal episodes after 48 hours. On day 3, glycopyrrolate (0.02 mg/kg IM) was administered with seeming reduction but not resolution of the episodes. Small amounts of feed (half of normal feeding amount) were offered at 6-hour intervals and clinical signs began to resolve. Feedings were continued under observation and syncopal events occurred once a day for 14 days, after which no further syncopal events were observed. n nAlthough swallow syncope has been reported in people,1,2 we are unaware of reports in animals. Typically syncope is related to deglutition (food or liquid), is often accompanied by bradyarrhythmias after swallowing leading to cerebral hypoperfusion and unconsciousness. The pathophysiology of swallow syncope is incompletely understood. Swallow syncope has been associated with organic or functional disorders of the esophagus (spasm, achalasia, etc.), cardiac disease (sinus bradycardia, sinoatrial or complete AV block, atrial fibrillation), and after thoracic surgery with transient hypoxia.3 However, in most human cases an underlying disease is not identified. Vagal reflex plays a major role because of the common innervation of the esophagus and heart in people, sheep, and pigs. Efferent signals lead to bradyarrhythmia and a temporary decrease in cardiac output with cerebral hypoperfusion. Management usually involves withdrawal of medications like beta-blockers that can cause a delay in cardiac conduction and vasodilatation, and administration of anticholinergics like atropine used to prevent bradyarrhythmias. n nDiagnosis of swallow syncope requires demonstration of a direct relationship between swallowing and syncope. Our sheep had no bradyarrhythmias on ECG monitoring (continuous for first 14 hours and twice daily after that) and postoperative echocardiography showed normal heart valve function with a good ejection fraction. Swallowing syncope started several hours after cardiac surgery and episodes occurred spontaneously when swallowing while the sheep was relaxed. Esophageal abnormalities and impairment of the sinoatrial or atrioventricular nodes were not identified. An esophageal motility disorder, a known side effect of thoracic or cardiac surgery, may have mediated syncope. One possible explanation for swallow syncope is that afferent sensory fibers may have artificially synapsed with efferent vagal fibers to the heart, producing a parasympathetic vagal impulse from an afferent sensory process like swallowing.1 n nSwallow syncope has not been reported in human surgical cases where fentanyl was used for anesthesia; however vasovagal syncopal episodes, decreased peripheral vascular resistance, and cardiovascular effects of opioids have been reported in people and animals.4–7 Naloxone was administered to this sheep in the immediate postoperative period because it was suspected that a substantial amount of fentanyl (continuous infusion for 6.5 hours at 0.1 µg/kg/min) used during surgery may have contributed to the onset of the syncopal events. n nSwallow syncope should not be considered a benign diagnosis and, depending on the history, animals should be investigated to rule out underlying esophageal or cardiac disease, including autonomic testing while eating.

Gene Therapy in Heart Failure.

Heart failure is a significant burden to the global healthcare system and represents an underserved market for new pharmacologic strategies, especially therapies which can address root cause myocyte dysfunction. Modern drugs, surgeries, and state-of-the-art interventions are costly and do not improve survival outcome measures. Gene therapy is an attractive strategy, whereby selected gene targets and their associated regulatory mechanisms can be permanently managed therapeutically in a single treatment. This in theory could be sustainable for the patients life. Despite the promise, however, gene therapy has numerous challenges that must be addressed together as a treatment plan comprising these key elements: myocyte physiologic target validation, gene target manipulation strategy, vector selection for the correct level of manipulation, and carefully utilizing an efficient delivery route that can be implemented in the clinic to efficiently transfer the therapy within safety limits. This chapter summarizes the key developments in cardiac gene therapy from the perspective of understanding each of these components of the treatment plan. The latest pharmacologic gene targets, gene therapy vectors, delivery routes, and strategies are reviewed.