Intramuscular injection of plasmid is a potential alternative to viral vectors for the transfer of therapeutic genes into skeletal muscle fibers. The low efficiency of plasmid-based gene transfer can be enhanced by electroporation (EP) coupled with the intramuscular application of hyaluronidase. We have investigated several factors that can influence the efficiency of plasmid-based gene transfer. These factors include electrical parameters of EP, optimal use of hyaluronidase, age and strain of the host, and plasmid size. Muscles of very young and mature normal, mdx, and immunodeficient mice were injected with plasmids expressing beta-galactosidase, microdystrophin, full-length dystrophin, or full-length utrophin. Transfection efficiency, muscle fiber damage, and duration of transgene expression were analyzed. The best transfection level with the least collateral damage was attained at 175-200 V/cm. Pretreatment with hyaluronidase markedly increased transfection, which was also influenced by the plasmid size and the strain and the age of the mice. Even in immunodeficient mice, there was a significant late decline in transgene expression and plasmid DNA copies, although both still remained relatively high after 1 year. Thus, properly optimized EP-assisted plasmid-based gene transfer is a feasible, efficient, and safe method of gene replacement therapy for dystrophin deficiency of muscle but readministration may be necessary.

HSPB7 belongs to the small heat-shock protein (sHSP) family, and its expression is restricted to cardiac and skeletal muscles from embryonic stages to adulthood. Here, we found that skeletal-muscle-specific ablation of the HspB7 does not affect myogenesis during embryonic stages to postnatal day 1 (P1), but causes subsequent postnatal death owing to a respiration defect, with progressive myopathy phenotypes in the diaphragm. Deficiency of HSPB7 in the diaphragm muscle resulted in muscle fibrosis, sarcomere disarray and sarcolemma integrity loss. We identified dimerized filamin C (FLNC) as an interacting partner of HSPB7. Immunofluorescence studies demonstrated that the aggregation and mislocalization of FLNC occurred in the muscle of HspB7 mutant adult mice. Furthermore, the components of dystrophin glycoprotein complex, γ- and δ-sarcoglycan, but not dystrophin, were abnormally upregulated and mislocalized in HSPB7 mutant muscle. Collectively, our findings suggest that HSPB7 is essential for maintaining muscle integrity, which is achieved through its interaction with FLNC, in order to prevent the occurrence and progression of myopathy.

Peripheral arterial tonometry (PAT) is a useful method to assess endothelial function. Analysis of pulse wave amplitude with PAT during reactive hyperemia can be used to study peripheral vascular endothelial function very well. We had injected a specific nitric oxide (NO) synthesize inhibitor intra-arterially into five adult male rats to verify the validation of our proposed photoplethysmograpy (PPG) system for endothelial function assessment. However, the reproducibility was not good for the system applied for measuring fingertip peripheral arteries. Therefore, an air-pressure sensing system with the high reproducibility was developed for peripheral vascular endothelial function assessment. This study showed that the vasodilatation index measured with air-pressure sensing system correlated very well with that measured using peripheral arterial tonometry. In addition, only few minutes are needed for conducting a self endothelial function assessment at home. And, early self-monitoring of cardiovascular dysfunction and arterial stiffness can be easily and effectively achieved.

Helper-dependent adenovirus vector (AdV)-mediated full-length dystrophin expression leads to significant mitigation of the dystrophic phenotype of the mdx mouse. However, dystrophin, as a neoantigen, elicits antibody formation. As an alternative approach, we evaluated gene transfer of full-length murine utrophin, a functional homologue of dystrophin that is normally present only at the neuromuscular junction. A single injection in the tibialis anterior (TA) muscle of the helper-dependent adenovirus vector encoding utrophin provided very good transduction, with 58% of fibers demonstrating sarcolemmal utrophin expression in the neonates, and 35% utrophin-positive (Utr(+)) fibers in adults. The presence of utrophin prevented extensive necrosis in the neonates, halted further necrosis in the adults, and led to restoration of sarcolemmal expression of dystrophin-associated proteins up to 1 year after injection. Marked physiological improvement was observed in both neonates and adults. Neither increased humoral responses nor cellular immune responses were evident. However, there was a time-related decline of the initial high utrophin expression. Although viral DNA persisted in animals that were injected in the neonatal stage, viral DNA levels decreased in muscles of adult mice. These results demonstrate that although utrophin gene transfer leads to amelioration of the dystrophic phenotype, the effects are not sustained upon loss of utrophin expression.

Physiological signals often show complex fluctuation (CF) under the dual influence of temporal and spatial scales, and CF can be used to assess the health of physiologic systems in the human body. This study applied multiscale cross-approximate entropy (MC-ApEn) to quantify the complex fluctuation between R-R intervals series and photoplethysmography amplitude series. All subjects were then divided into the following two groups: healthy upper middle-aged subjects (Group 1, age range: 41–80 years, n = 27) and upper middle-aged subjects with type 2 diabetes (Group 2, age range: 41–80 years, n = 24). There are significant differences of heart rate variability, LHR, between Groups 1 and 2 (1.94 ± 1.21 versus 1.32 ± 1.00, P = 0.031). Results demonstrated differences in sum of large scale MC-ApEn (MC-ApEnLS) (5.32 ± 0.50 versus 4.74 ± 0.78, P = 0.003). This parameter has a good agreement with pulse-pulse interval and pulse amplitude ratio (PAR), a simplified assessment for baroreflex activity. In conclusion, this study employed the MC-ApEn method, integrating multiple temporal and spatial scales, to quantify the complex interaction between the two physical signals. The MC-ApEnLS parameter could accurately reflect disease process in diabetics and might be another way for assessing the autonomic nerve function.

This study proposed a dynamic pulse wave velocity (PWV)-based biomedical parameter in assessing the degree of atherosclerosis for the aged and diabetic populations. Totally, 91 subjects were recruited from a single medical institution between July 2009 and October 2010. The subjects were divided into four groups: young healthy adults (Group 1, n = 22), healthy upper middle-aged adults (Group 2, n = 28), type 2 diabetics with satisfactory blood sugar control (Group 3, n = 21), and unsatisfactory blood sugar control (Group 4, n = 20). A self-developed six-channel electrocardiography (ECG)-PWV-based equipment was used to acquire 1000 successive recordings of PWV(foot) values within 30 min. The data, thus, obtained were analyzed with multiscale entropy (MSE). Large-scale MSE index (MEI(LS)) was chosen as the assessment parameter. Not only did MEI(LS) successfully differentiate between subjects in Groups 1 and 2, but it also showed a significant difference between Groups 3 and 4. Compared with the conventional parameter of PWV(foot) and MEI on R-R interval [i.e., MEI(RRI)] in evaluating the degree of atherosclerotic change, the dynamic parameter, MEI(LS) (PWV), could better reflect the impact of age and blood sugar control on the progression of atherosclerosis.

Dystrophin gene transfer using gutted or helper-dependent adenoviruses (HDAd), which have most of their genes deleted, is a promising approach to treat Duchenne muscular dystrophy. In an attempt to boost the amount of dystrophin produced after gene transfer, we have constructed a fully deleted HDAd (HDCBDys2x) containing two human dystrophin cDNAs controlled by the powerful hybrid cytomegalovirus enhancer/beta-actin promoter. We demonstrated high dystrophin expression after infection of muscle cultures with HDCBDys2x. Similarly, high (mean=583) and moderate (mean=124) numbers of muscle fibers were transduced in anterior tibialis muscle after intramuscular injection of HDCBDys2x in neonate and adult dystrophindeficient (mdx) mice 10 days postinjection. In fact, in the neonatally injected mdx mice, the transferred dystrophin was five times more abundant than in normal human muscle. However, the high early transduction level was transient in both animal groups, and we observed a humoral response against the human dystrophin. In contrast, we demonstrated sustained dystrophin expression in immunodeficient mouse muscles. Dystrophin expression of HDCBDys2x could be further increased in the presence of an E1/E3-deleted (first-generation) adenovirus, thus demonstrating that the latter vector synthesizes trans-acting enhancing factors. We have achieved abundant dystrophin expression with our new, improved HDAd. It is anticipated that high longterm transgene expression will be possible by employing weaker immunogenic transgenes.

Pulse wave velocity (PWV) is a useful method to assess arterial stiffness and predict mortality of atherosclerosis-related diseases. The progression of atherosclerosis is not homogeneous. There must be difference of PWV between sites by site. Therefore we designed a multi-channel instrument to measure PWV at different sites of the body simultaneously. We measured PWV at six different regions simultaneously. Thirty four healthy adults received the measurement. We found that PWVs were higher in the large vessels, by measuring from the heart to toes and heart to fingers as compared with the measurement from the heart to the earlobes (4.76 ± 0.46 m/s; 4.67 ± 0.41 m/s; 1.10 ± 0.16 m/s). The PWV of the left and right sides were the same. Although there were statistically significances, the correlation of PWV between foot and hand is better than those between ear and foot and between ear and hand. Herein we presented a novel and reliable measurement of PWV. The changes of PWV in different regions may be used in predicting disease processes such as stroke, coronary artery diseases and renal diseases, respectively.