Abstract-CalciumKey Words: hypertrophy Ⅲ calcium Ⅲ gene therapy C alcium cycling in the heart is triggered by calcium influx through L-type calcium channels. 1 Such calcium channels are present and functionally important not only in cardiac myocytes but also in diverse smooth muscles and in neurons. Enhancement of calcium-regulated signaling pathways contributes to the development of left ventricular hypertrophy (LVH). 2 Thus, calcium channel inhibition represents a logical approach to treatment of LVH. Gene therapy allows for more directed and organ-specific delivery and thus may avoid undesired systemic effects, which may account in part for the limited clinical benefit observed with these agents. 3 L-type calcium channels are heteromultimers of various subunits. The accessory ␤ subunit (LTCC␤) not only favors the trafficking of the calcium channel to the surface membrane, but also enhances the probability of channel opening 4 -7 resulting in increased calcium current. Interestingly, upregulation of a splice variant of the LTCC␤ (␤ 2a ) has been previously described in failing human cardiomyocytes. 8 Whether or not ␤ subunits are upregulated in hypertrophy, we hypothesized that suppression of LTCC␤ might represent an attractive means to inhibit calcium influx, attenuate calciumdependent signaling and ultimately prevent or treat LVH.Using RNA interference technology to selectively modulate the expression of the gene of interest, we studied the physiological effects of LTCC␤ modulation in native cardiac cells, and the effects in a cellular model of hypertrophy. To further investigate the effects of LTCC␤ downregulation in cardiac hypertrophy, we performed in vivo gene transfer of an "advanced" generation lentiviral vector capable of permanently modulating the expression of LTCC␤. A rat pressureoverload LVH model was implemented to test the potential beneficial effect of modulating the expression of the L-type calcium channel accessory ␤ subunit. Materials and MethodsFor a detailed Materials and Methods, please see the online data supplement available at http://circres.ahajournals.org. Short Hairpin RNA Design and Vector ProductionA series of short interference RNA duplexes (siRNAs) against the D2 conserved domain 9 of LTCC␤ were designed according to published algorithms 10 and synthesized. The three most active sequences and one scrambled, nonsilencing (NS) sequence were designed into a short hairpin RNA (shRNA) oligonucleotide. These three active shRNAs and one NS-shRNA sequence were screened Original

Seminal to the process of arterial restenosis after balloon angioplasty is extracellular matrix degradation by metalloproteinases (MMPs); activity of these proteins is strongly inhibited by the tissue inhibitors of MMPs (TIMPs). Here we exploit gene transfer using an adeno-associated virus (AAV) for TIMP1 gene delivery in a rat model of intimal hyperplasia. High-titer AAV-Timp1 efficiently transduced human coronary artery smooth muscle cells (SMCs) in vitro and inhibited the capacity of these cells to migrate through a Matrigel barrier. In injured rat carotid arteries, AAV vectors were found to transduce SMCs efficiently and to maintain transgene expression for several weeks in vivo. In AAV-Timp1-transduced animals, the intima:media ratio of injured carotids was significantly reduced by 70.5% after 2 weeks, by 58.5% after 1 month, and by 52.4% after 2 months from treatment. The decrease in intimal hyperplasia was paralleled by a significant inhibition of collagen accumulation and by increased elastin deposition in the neointima, two findings that relate to the inhibition of MMP activity. These results indicate that AAV vectors are efficient tools for delivering genes to the arterial wall and emphasize the importance of MMPs for the generation of intimal hyperplasia. Local TIMP1 gene transfer might thus represent an efficient strategy to prevent restenosis.

In failing hearts, the force-frequency response (FFR) is blunted, flat or negative. A positive FFR is crucial for healthy myocardium to respond to an increased working demand. There is no consensus in weather a positive FFR relies on myofilament Ca 2+ sensitization or desensitization and weather this is modulated by cTnI phosphorylation. In the present work we aimed to address the FFR and Ca 2+ cycling in intact mouse trabeculae loaded with Fura-2. To achieve this we used two transgenic models with pseudo phosphorylation mutants of troponin I (TnI), TnIDD 22,23 mice, which mimic increased phosphorylation at PKA sites of TnI at Ser 22 and 23 and TnI PKA/PKC mice, which mimic dephosphorylation at same PKA sites and increased phosphorylation at PKC sites of TnI at Ser 42 and 44. We hypothesized that controlling for cTnI phosphorylation will clarify the contribution of cTnI to the differences in force and Ca 2+ dynamics during FFR. When we examined the isometric contraction and Ca 2+ dynamics in each of these lines (TnIDD 22,23 , n= 8; TnI PKA/PKC, n=6) and non transgenic controls (NTG, n=7) we found that all three groups showed a positive FFR, although peak Ca 2+ increased with frequency rate in all three a less steep Ca 2+ transient increase (myofilament Ca 2+ sensitization) was observed in both transgenic lines compared to NTG (TnIDD 22,23 , p= 0.001; TnI PKA/PKC, p=0.03). Additionally, the peak force during the FFR was greater in the TnIDD 22,23 mice compared to NTG (p < 0.0001), suggesting that TnIDD 22,23 mice posses an enhanced frequency rate-related myofilament Ca 2+ sensitivity. WB analysis of Ca 2+ handling proteins including PLB, pPLB, SERCA2a and Ryanodine receptor normalized levels showed no major differences among all three groups, suggesting the differences observed in TnIDD 22,23 mice were not due to altered Ca 2+ handling but rather to myofilament Ca 2+ sensitivity. We conclude that a positive systolic peak FFR is followed by increasing myofilament Ca 2+ esensitization but mimicking increased phosphorylation at PKA sites of TnI Ser 22,23 enhances FFR and Ca 2+ responsiveness. Overall, our results support the concept that myofilament alterations feedback onto Ca 2+ handling mechanisms and these findings have important implications for human heart failure.

Recently, we have identified specific sites of O -GlcNAcylation in major cardiac myofilament proteins. We used a methodology based on GalNaz-Biotin labeling followed by DTT switch and LC-MS/MS site mapping. As a result, 42 O- GlcNAc peptides from cardiac myofilaments were identified, corresponding to 32 of cardiac myosin heavy chain (MHC), 6 of α-sarcomeric actin, 2 of myosin light chain 1 (MLC1), 1 of MLC2, and 1 of troponin I (cTnI). Most of the identified O -GlcNAcylation sites are novel post-translational modification sites. To assess the potential physiological role of myofilament GlcNAcylation, Force-Calcium relationships studies were performed on skinned rat trabeculae. We have previously reported that exposure to GlcNAc but not Glycerol significantly decreases calcium sensitivity, further investigation confirm these preliminary findings (pCa 50 1.81 ± 0.13 μM for Control vs. 3.83 ± 0.44 μM for GlcNAc, n =7, P <=.001), and in addition demonstrate that maximal force (F max ) and Hill coefficient ( n ) are not significantly changed. Troponin I phosphorylation at Ser23, 24 was determined in three pooled trabeculea by WB using a specific antibody phosphor-TnI (Cell Signaling) normalized to actin signal (GlcNAc 0.722 vs Glycerol 0.667 A.U., n=3, p=NS). Phosphorylation at this PKA sites was ruled out as responsible for myofilament desensitization. Acute exposure of cardiac myofilaments to GlcNAc significantly increased α-sarcomeric actin O -GlcNAcylation from 27.6±4.2% to 35.1±2.36% ( n = 4, p<0.05) whereas total GlcNAcylation levels were unchanged. Additionally, we showed by immunofluoresence that O-GlcNAc transferase (OGT) and O-GlcNAcase (OGase) are abundant in rat hearts, OGT localizes predominantly in cardiac nuclei and less in cytoplasm, whereas OGase shows the opposite pattern. These studies provides the first site mapping of O -GlcNAcylation sites in cardiac myofilament proteins, and demonstrates their potential role in regulating myocardial contractile function. Regulation of myofilament O -GlcNAcylation may represent a novel and useful therapeutic target in heart failure, especially in diabetic cardiomyopathy.