Oliver Y. Bernecker

PICOT Inhibits Cardiac Hypertrophy and Enhances Ventricular Function and Cardiomyocyte Contractility

Multiple signaling pathways involving protein kinase C (PKC) have been implicated in the development of cardiac hypertrophy. We observed that a putative PKC inhibitor, PICOT (PKC-Interacting Cousin Of Thioredoxin) was upregulated in response to hypertrophic stimuli both in vitro and in vivo. This suggested that PICOT may act as an endogenous negative feedback regulator of cardiac hypertrophy through its ability to inhibit PKC activity, which is elevated during cardiac hypertrophy. Adenovirus-mediated gene transfer of PICOT completely blocked the hypertrophic response of neonatal rat cardiomyocytes to enthothelin-1 and phenylephrine, as demonstrated by cell size, sarcomere rearrangement, atrial natriuretic factor expression, and rates of protein synthesis. Transgenic mice with cardiac-specific overexpression of PICOT showed that PICOT is a potent inhibitor of cardiac hypertrophy induced by pressure overload. In addition, PICOT overexpression dramatically increased the ventricular function and cardiomyocyte contractility as measured by ejection fraction and end-systolic pressure of transgenic hearts and peak shortening of isolated cardiomyocytes, respectively. Intracellular Ca2+ handing analysis revealed that increases in myofilament Ca2+ responsiveness, together with increased rate of sarcoplasmic reticulum Ca2+ reuptake, are associated with the enhanced contractility in PICOT-overexpressing cardiomyocytes. The inhibition of cardiac remodeling by of PICOT with a concomitant increase in ventricular function and cardiomyocyte contractility suggests that PICOT may provide an efficient modality for treatment of cardiac hypertrophy and heart failure.

Apoptosis in heart failure and the senescent heart

The progressive loss of cardiac myocytes by apoptotic cell death has been discussed as an important pathogenic component in the failing myocardium as well in the aging heart. The degree to which apoptosis contributes to myocyte loss in these conditions, however, is a controversial issue. This review focuses on the regulation of apoptosis, evidence implicating apoptosis as a mechanism for the progression and development of heart failure, the role of apoptotic death in senescent cardiac dysfunction, as well as on the problems of detection of apoptosis.

Increased leakage of sarcoplasmic reticulum Ca2+ contributes to abnormal myocyte Ca2+ handling and shortening in sepsis

Objective:Changes in cardiac function due to sepsis have been widely reported. However, the underlying mechanisms remain poorly understood. In the mammalian heart, myocyte function and intracellular calcium homeostasis are closely coupled. In this study we tested the hypothesis that alterations in cardiac calcium homeostasis due to sepsis underlie the observed myocyte dysfunction. Design:Randomized prospective animal study. Setting:Research laboratory. Subjects:Male Sprague-Dawley rats weighing 250–275 g. Interventions:We induced sepsis by cecal ligation and puncture in the rat, which mimics the type of infection caused by perforation of the intestine in humans. Measurements and Results:Forty-eight hours after cecal ligation and puncture, isolated cardiac ventricular cardiomyocytes demonstrated a 57% decreased peak systolic [Ca2+]. The time constant of the Ca2+ transient increased 71% and 57% in myocytes obtained 24 hrs and 48 hrs after cecal ligation and puncture, respectively. The average shortening of cardiomyocytes 48 hrs after cecal ligation and puncture was significantly decreased. To investigate the cellular mechanisms of altered Ca2+ transients and myocyte shortening, we measured Ca2+ sparks, the spontaneous local Ca2+ release events in cardiomyocytes at resting states. The Ca2+ spark frequency progressively increased in myocytes 24 hrs and 48 hrs after cecal ligation and puncture. The total activity of sparks also increased compared with sham-operated animals. The overall leakage of sarcoplasmic reticulum Ca2+ in resting states was increased in sepsis and resulted in reduced sarcoplasmic reticulum Ca2+ content. Conclusions:Abnormal Ca2+ leakage from the sarcoplasmic reticulum contributes significantly to the depressed myocyte shortening in sepsis. In the future, modalities that prevent this Ca2+ leakage may prove beneficial in the treatment of sepsis-induced myocyte shortening.

Cardiac-Specific Gene Expression Facilitated by an Enhanced Myosin Light Chain Promoter

BACKGROUND Adenoviral gene transfer has been shown to be effective in cardiac myocytes in vitro and in vivo. A major limitation of myocardial gene therapy is the extracardiac transgene expression. METHODS To minimize extracardiac gene expression, we have constructed a tissue-specific promoter for cardiac gene transfer, namely, the 250-bp fragment of the myosin light chain-2v (MLC-2v) gene, which is known to be expressed in a tissue-specific manner in ventricular myocardium followed by a luciferase (luc) reporter gene (Ad.4 x MLC250.Luc). Rat cardiomyocytes, liver and kidney cells were infected with Ad.4 x MLC.Luc or control vectors. For in vivo testing, Ad.4 x MLC250.Luc was injected into the myocardium or in the liver of rats. Kinetics of promoter activity were monitored over 8 days using a cooled CCD camera. RESULTS In vitro: By infecting hepatic versus cardiomyocyte cells, we found that the promoter specificity ratio (luc activity in cardiomyocytes per liver cells) was 20.4 versus 0.9 (Ad.4 x MLC250.Luc vs. Ad.CMV). In vivo: Ad.4 x MLC250.Luc significantly reduced luc activity in liver (38.4-fold), lung (16.1-fold), and kidney (21.8-fold) versus Ad.CMV (p =.01); whereas activity in the heart was only 3.8-fold decreased. The gene expression rate of cardiomyocytes versus hepatocytes was 7:1 (Ad.4 x MLC.Luc) versus 1:1.4 (Ad.CMV.Luc). DISCUSSION This new vector may be useful to validate therapeutic approaches in animal disease models and offers the perspective for selective expression of therapeutic genes in the diseased heart.

Diltiazem during reperfusion preserves high energy phosphates by protection of mitochondrial integrity

OBJECTIVE This study evaluates the effects of diltiazem administered during reperfusion on hemodynamic, metabolic, and ultrastructural postischemic outcome. METHODS Hearts of 38 adult White New Zealand rabbits underwent 60 min of global cold ischemia followed by 40 min of reperfusion in an erythrocyte perfused isolated working heart model. Hearts were randomly assigned to four groups and received diltiazem (0.1, 0.25, and 0.5 micromol/l) during reperfusion only, or served as control. RESULTS The postischemic time courses of heart rate, aortic flow, and external stroke work clearly reflected the dose-dependent negative chronotropic and inotropic efficacy of diltiazem in the two higher concentrations. High energy phosphates (HEP) determined from myocardial biopsies taken after 40 min of reperfusion were significantly better preserved in all treatment groups compared to control hearts. Similarly ultrastructural grading of mitochondria and myofilaments revealed a significant reduction of reperfusion injury in hearts that received diltiazem compared to control. CONCLUSIONS Diltiazem protects mitochondrial integrity and function, thereby preserving myocardial HEP levels. Only low dose diltiazem (0.1 micromol/l) during reperfusion combines both, optimal mitochondrial preservation with minimal changes in hemodynamics.

Gene therapy for the treatment of heart failure--calcium signaling.

The knowledge of molecular mechanisms indicated in cardiac dysfunction has increased dramatically over the last decade and yields considerable potential for new treatment options in heart failure. Alterations in intracellular calcium signaling play a crucial role in the pathophysiology of heart failure, and in recent years, somatic gene transfer has been identified as an important tool to help understand the relative contribution of specific calcium-handling proteins in heart failure. This article reviews recent advances in gene delivery techniques aimed at global myocardial transfection and discusses molecular therapeutic targets identified within intracellular calcium signaling pathways in heart failure.

872. Highly Efficient Virus-Mediated Gene Transfer to Human Vein Grafts Via Intraluminal Pressure Delivery

Top of pageAbstract Introduction Long term patency rates for coronary artery bypass grafting (CABG) using autologous saphenous vein are poor, showing 10 years post-CABG patency rates of 41%. To date, effective and proven pharmacologic interventions to prolong vein graft patency are lacking. Gene therapy seems particularly well suited for the prevention or postponement of vein graft failure since (1) the stimulation of smooth muscle cell proliferation appears to largely be an early and transient process, and (2) the target tissue is easily accessible ex vivo during the surgical procedure. However, a major obstacle in efficient gene transfer to human vein grafts is the heterogenity of vessel quality due to age, preexisting veinous disease, and surgical trauma. In this study we developed a clinically relevant viral delivery technique to achieve a reproducible and efficient gene transfer in human vein grafts. Methods 46 intact human saphenous veins (age 68+/|[minus]|7) from patients undergoing coronary bypass surgery were cut into rings 15 mm in length. A small piece of each vein was tested for endothelial viability with trypan blue. Individual vein rings were endoluminally incubated with adenoviral vectors expressing two reportergenes (Ad.CMV.lacZ/GFP, 1|[times]|10E11 pfu/mL) with different intraluminal pressures (50, 100, 150mmHg) for 30 or 60 minutes. Veinsegments were cultured for 6days after transfection and harvested for cryopreservation. Transfection efficiency was evaluated by X-gal staining and GFP- Westernblot; endothelial integrity and neointima-hyperplasia were evaluated by CD31-staining, Elastica Van Gieson-staining and H-E-staining. Results Veins which underwent endoluminal gene transfer without pressure showed only poor transfection efficiency (12%+/|[minus]|7). In contrast, veins wich were pressure-transfected with either 100 or 150 mmHg showed high transfection efficiency throughout the vessel wall (75%|[minus]|+/|[minus]|13 and 91+/|[minus]|7% respectively, p<0.02). Veins transfected for 60 minutes showed no significant difference in transfection efficiency versus 30 minute incubation. However, CD31 staining revealed a significant loss of endothelium in the high pressure group (150 mmHg), whereas the 100 mmHg pressure group showed only minor endothelial damage compared to control veins, irrespective of incubation time. Discussion This study presents an optimized transfection protocol for gene transfer in human vein grafts. We here show for the first time that viral transfection using carefully regulated supraphysiological endoluminal pressure greatly enhances transfection efficiency in human veins. Using continuous intraluminal pressure-monitoring and careful evaluation of endothelial integrity, we found optimal conditions for highly efficient and reproducible transfection using 100 mmHg intraluminal pressure for 30 minutes. Establishing a safe and efficient delivery method in a clinically relevant model for human cardiovascular gene therapy is an important aspect of translation from pre-clinical to clinical gene therapy.