Ngai Chin Lai

Comparison of Adeno-Associated Virus Serotypes and Delivery Methods for Cardiac Gene Transfer

Cardiac gene transfer is a potentially useful strategy for cardiovascular diseases. The adeno-associated virus (AAV) is a common vector to obtain transgene expression in the heart. Initial studies conducted in rodents used indirect intracoronary delivery for cardiac gene transfer. More recently AAV vectors with so-called cardiac tropism have enabled significant cardiac transgene expression following intravenous injection. However, a direct comparison of intravenous versus intracoronary delivery with rigorous quantification of cardiac transgene expression has not been conducted. In the present study we tested the hypothesis that intracoronary AAV delivery would be superior to intravenous delivery vis-à-vis cardiac transgene expression. We compared intravenous and intracoronary delivery of AAV5, AAV6, and AAV9 (5×10(11) genome copies per mouse). Using enhanced green fluorescent protein as a reporter, we quantified transgene expression by fluorescence intensity and Western blotting. Quantitative polymerase chain reaction (PCR) was also performed to assess vector DNA copies, employing primers against common sequences on AAV5, AAV6, and AAV9. Intracoronary delivery resulted in 2.6- to 28-fold higher transgene protein expression in the heart 3 weeks after AAV injection compared to intravenous delivery depending on AAV serotype. The highest level of cardiac gene expression was achieved following intracoronary delivery of AAV9. Intracoronary delivery of AAV9 is a preferred method for cardiac gene transfer.

Pressure overload-induced cardiac remodeling and dysfunction in the absence of interleukin 6 in mice.

Congestive heart failure is associated with increased expression of pro-inflammatory cytokines, and the levels of these cytokines correlate with heart failure severity and prognosis. Chronic interleukin 6 (IL-6) stimulation leads to left ventricular (LV) hypertrophy and dysfunction, and deletion of IL-6 reduces LV hypertrophy after angiotensin II infusion. In this study, we tested the hypothesis that IL-6 deletion has favorable effects on pressure-overloaded hearts. We performed transverse aortic constriction on IL-6-deleted (IL6KO) mice and C57BL/6J mice (CON) to induce pressure overload. Pressure overload was associated with similar LV hypertrophy, dilation, and dysfunction in CON and IL6KO mice. Re-activation of the fetal gene program was also similar in pressure-overloaded CON and IL6KO mice. There were no differences between CON and IL6KO mice in LV fibrosis or expression of extracellular matrix proteins after pressure overload. In addition, no group differences in apoptosis or autophagy were seen. These data indicate that IL-6 deletion does not block LV remodeling and dysfunction induced by pressure overload. Attenuated content of IL-11 appears to be a compensatory mechanism for IL-6 deletion in pressure-overloaded hearts. We infer from these data that limiting availability of IL-6 alone is not sufficient to attenuate LV remodeling and dysfunction in failing hearts.

Beneficial Effects of Adenylyl Cyclase Type 6 (AC6) Expression Persist Using a Catalytically Inactive AC6 Mutant

Cardiac-directed expression of AC6 has pronounced favorable effects on cardiac function possibly not linked with cAMP production. To determine rigorously whether cAMP generation is required for the beneficial effects of increased AC6 expression, we generated a catalytically inactive AC6 mutant (AC6mut) that has markedly diminished cAMP generating capacity by replacing aspartic acid with alanine at position 426 in the C1 domain (catalytic region) of AC6. Gene transfer of AC6 or AC6mut (adenovirus-mediated) in adult rat cardiac myocytes resulted in similar expression levels and intracellular distribution, but AC6mut expression was associated with marked reduction in cAMP production. Despite marked reduction in cAMP generation, AC6mut influenced intracellular signaling events similarly to that observed after expression of catalytically intact AC6. For example, both AC6 and AC6mut reduced phenylephrine-induced cardiac myocyte hypertrophy and apoptosis (p < 0.001), expression of cardiac ankyrin repeat protein (p < 0.01), and phospholamban (p < 0.05). AC6mut expression, similar to its catalytically intact cohort, was associated with increased Ca2+ transients in cardiac myocytes after isoproterenol stimulation. Many of the biological effects of AC6 expression are replicated by a catalytically inactive AC6 mutant, indicating that the mechanisms for these effects do not require increased cAMP generation.

Preserved cardiac function despite marked impairment of cAMP generation.

Objectives So many clinical trials of positive inotropes have failed, that it is now axiomatic that agents that increase cAMP are deleterious to the failing heart. An alternative strategy is to alter myocardial Ca2+ handling or myofilament response to Ca2+ using agents that do not affect cAMP. Although left ventricular (LV) function is tightly linked to adenylyl cyclase (AC) activity, the beneficial effects of AC may be independent of cAMP and instead stem from effects on Ca2+ handling. Here we ask whether an AC mutant molecule that reduces LV cAMP production would have favorable effects on LV function through its effects on Ca2+ handling alone. Methods and Results We generated transgenic mice with cardiac-directed expression of an AC6 mutant (AC6mut). Cardiac myocytes showed impaired cAMP production in response to isoproterenol (74% reduction; p<0.001), but LV size and function were normal. Isolated hearts showed preserved LV function in response to isoproterenol stimulation. AC6mut expression was associated with increased sarcoplasmic reticulum Ca2+ uptake and the EC50 for SERCA2a activation was reduced. Cardiac myocytes isolated from AC6mut mice showed increased amplitude of Ca2+ transients in response to isoproterenol (p = 0.0001). AC6mut expression also was associated with increased expression of LV S100A1 (p = 0.03) and reduced expression of phospholamban protein (p = 0.01). Conclusion LV AC mutant expression is associated with normal cardiac function despite impaired cAMP generation. The mechanism appears to be through effects on Ca2+ handling — effects that occur despite diminished cAMP.