Chronic suppression of heart-failure progression by a pseudophosphorylated mutant of phospholamban via in vivo cardiac rAAV gene delivery.

The feasibility of gene therapy for cardiomyopathy, heart failure and other chronic cardiac muscle diseases is so far unproven. Here, we developed an in vivo recombinant adeno-associated virus (rAAV) transcoronary delivery system that allows stable, high efficiency and relatively cardiac-selective gene expression. We used rAAV to express a pseudophosphorylated mutant of human phospholamban (PLN), a key regulator of cardiac sarcoplasmic reticulum (SR) Ca(2+) cycling in BIO14.6 cardiomyopathic hamsters. The rAAV/S16EPLN treatment enhanced myocardial SR Ca(2+) uptake and suppressed progressive impairment of left ventricular (LV) systolic function and contractility for 28-30 weeks, thereby protecting cardiac myocytes from cytopathic plasma-membrane disruption. Low LV systolic pressure and deterioration in LV relaxation were also largely prevented by rAAV/S16EPLN treatment. Thus, transcoronary gene transfer of S16EPLN via rAAV vector is a potential therapy for progressive dilated cardiomyopathy and associated heart failure.

Chronic phospholamban inhibition prevents progressive cardiac dysfunction and pathological remodeling after infarction in rats.

Ablation or inhibition of phospholamban (PLN) has favorable effects in several genetic murine dilated cardiomyopathies, and we showed previously that a pseudophosphorylated form of PLN mutant (S16EPLN) successfully prevented progressive heart failure in cardiomyopathic hamsters. In this study, the effects of PLN inhibition were examined in rats with heart failure after myocardial infarction (MI), a model of acquired disease. S16EPLN was delivered into failing hearts 5 weeks after MI by transcoronary gene transfer using a recombinant adeno-associated virus (rAAV) vector. In treated (MI-S16EPLN, n = 16) and control (MI-saline, n = 18) groups, infarct sizes were closely matched and the left ventricle was similarly depressed and dilated before gene transfer. At 2 and 6 months after gene transfer, MI-S16EPLN rats showed an increase in left ventricular (LV) ejection fraction and a much smaller rise in LV end-diastolic volume, compared with progressive deterioration of LV size and function in MI-saline rats. Hemodynamic measurements at 6 months showed lower LV end-diastolic pressures, with enhanced LV function (contractility and relaxation), lowered LV mass and myocyte size, and less fibrosis in MI-S16EPLN rats. Thus, PLN inhibition by in vivo rAAV gene transfer is an effective strategy for the chronic treatment of an acquired form of established heart failure.

The antioxidant N-2-mercaptopropionyl glycine attenuates left ventricular hypertrophy in in vivo murine pressure-overload model.

OBJECTIVES: In order to identify the role of reactive oxygen species (ROS) in cardiac hypertrophy, we examined the effect of N-2-mercaptopropionyl glycine (MPG) on cardiac hypertrophy. BACKGROUND: Recent in vitro studies have suggested that ROS play an important role as a second messenger in cardiac hypertrophy. It was therefore thought to be of particular value to examine the relevance of studies using in vitro models for cardiac hypertrophy in an in vivo setting. METHODS: The transverse thoracic aorta in mice was constricted, and MPG (100 mg/kg) was infused intraperitoneally twice daily. The animals were assessed seven days after the operation for hemodynamic functions, oxidative stress and antioxidative enzyme activities. RESULTS: Banding of the transverse aorta in mice resulted in an increase in the ratio of heart weight to tibia length and the appearance of an endogenous atrial natriuretic factor messenger ribonucleic acid (mRNA) seven days postoperatively. Administration of MPG significantly attenuated the hypertrophic responses induced by pressure overload. Cardiac hypertrophy was accompanied by increases in heme oxygenase-1 mRNA expression and lipid peroxidation, which was eliminated by the treatment with MPG. Pressure overload led to increases in antioxidant enzyme activities, such as superoxide dismutase and glutathione peroxidase, but not catalase, activity. CONCLUSIONS: Our results indicated that oxidative stress was increased in our model and that it plays an important role in the development of cardiac hypertrophy.

Cardiac-specific overexpression of a high Ca2+ affinity mutant of SERCA2a attenuates in vivo pressure overload cardiac hypertrophy.

In cardiomyocytes, calcium plays important roles as a signal in cardiac hypertrophy and contraction-relaxation cycling. Elevation of Ca2+ concentration in myoplasm is associated with the onset and progression of hypertrophy as well as the enhancement of contractility. The cardiac Ca2+ ATPase (SERCA2a) of the sarcoplasmic reticulum plays a dominant role in lowering cytoplasmic calcium levels during relaxation and is regulated by phospholamban (PLN). To examine whether the modulation of SERCA2a activity results in the attenuation of cardiac hypertrophy and enhancement of contractility, we generated transgenic mice (TG) overexpressing a high calcium affinity SERCA2a mutant (K397/400E), lacking a functional association with PLN. In the TG hearts, the apparent affinity of SERCA2a for Ca2+ significantly increased compared with their nontransgenic littermate controls. The TG showed increased contraction and relaxation, with increases in the amplitude of Ca2+ transient and rapid Ca2+ decay. Upon induction of pressure overload by transverse aortic constriction, the TG developed less cardiac hypertrophy than littermate controls did. The activation of Ca2+-sensitive protein kinase C by pressure overload was significantly attenuated in the TG hearts. Our findings indicate an association of SERCA2a activity with cardiac hypertrophy and thus a new therapeutic target for the prevention and treatment of cardiac hypertrophy.

Young MLP deficient mice show diastolic dysfunction before the onset of dilated cardiomyopathy.

Targeted deletion of cytoskeletal muscle LIM protein (MLP) in mice consistently leads to dilated cardiomyopathy (DCM) after one or more months. However, next to nothing is known at present about the mechanisms of this process. We investigated whether diastolic performance including passive mechanics and systolic behavior are altered in 2-week-old MLP knockout (MLPKO) mice, in which heart size, fractional shortening and ejection fraction are still normal. Right ventricular trabeculae were isolated from 2-week-old MLPKO and wildtype mice and placed in an apparatus that allowed force measurements and sarcomere length measurements using laser diffraction. During a twitch from the unloaded state at 1 Hz, MLPKO muscles relengthened to slack length more slowly than controls, although the corresponding force relaxation time was unchanged. Active developed stress at a diastolic sarcomere length of 2.00 microm was preserved in MLPKO trabeculae over a wide range of pacing frequencies. Force relaxation under the same conditions was consistently prolonged compared with wildtype controls, whereas time to peak and maximum rate of force generation were not significantly altered. Ca2+ content of the sarcoplasmic reticulum (SR) and the quantities of Ca2+ handling proteins were similar in both genotypes. In summary, young MLPKO mice revealed substantial alterations in passive myocardial properties and relaxation time, but not in most systolic characteristics. These results indicate that the progression to heart failure in the MLPKO model may be driven by diastolic myocardial dysfunction and abnormal passive properties rather than systolic dysfunction.