Duchenne Muscular Dystrophy (DMD) is a degenerative disease leading to fatal respiratory and cardiac complications. It is caused by loss of functional dystrophin through mutation of the dystrophin gene. Possible treatments could use Adeno-associated virus (AAV) to introduce genes into muscle cells. This strategy requires muscle specific regulatory cassettes containing sequences specific for transcribing the therapeutic gene in muscle cells. My studies seek to increase the activity of one such cassette, MHCK7. Since AAV vectors can package only ~5000 basepairs of DNA, the dystrophin cDNA and regulatory cassette must be miniaturized. I am modifying MHCK7 by adding a 95 bp intronic enhancer (SIE) from the muscle creatine kinase (MCK) gene that is important for MCK expression in slow muscle fibers and cardiac muscle. The SIE has been placed ~1kb 3’ of the MHCK7 luciferase reporter cDNA to mimic its endogenous genomic location. SIE addition was performed by introducing a HindIII site 3’ of the luciferase polyA sequence by site directed mutagenesis, and the subsequent opening of DNA at that site. The SIE was transferred from a previously made plasmid and ligated into the opened MHCK7. Restriction digests and gel electrophoresis were used to assess successful introduction of HindIII and to confirm digests for opening the DNA and isolating the SIE. New recombinant MHCK7-3’-SIE will be transfected into skeletal and cardiac muscle cultures, and its activity will be assessed through luciferase activity. The SIE’s contribution to expression will be compared to that of the original MHCK7 plasmid as well as to MHCK7 with the SIE located immediately 5’ of the cassette. If the new regulatory cassette is more active than MHCK7 and 5’-SIE-MHCK7, further studies will test the cassette in conjunction with a microdystrophin cDNA. If activity is not increased, I will test alternative locations of the SIE.

Approximately 1 million Americans live with a lower-limb amputation and this number is increasing. During both the postoperative recovery period (up to 18 months) and the subsequent period patients undergo substantial changes in the volume and shape of their residual limb. Therefore, both the fit and comfort of their prosthetic are compromised. Edema, gait instability and skin irritation are often severe consequences. Patients often use socks, pads and fluid-filled bladders to compensate for these changes in volume. I am currently investigating the fluctuations in the volume of the residual limb of below-the-knee (trans-tibital) amputees. The goal is to identify patterns in the volume fluctuations over the course of the day. We have created sock logs on which patients record the changes in their sock ply during the day. Our goal is to provide practitioners, patients, and industry with insight into how often and when patients change their sock ply. We hope, with this knowledge, to propose more automated solutions for patients to manage their residual limb volume. Automation is needed because many patients elect not to change sock ply even though they need to so as to maintain a proper prosthetic fit. Similarly, our research is also focused on investigating the correlation between limb volume changes and the need for a new socket. With this data, we hope to establish a relationship between the changes in sock ply and the time at which a new prosthetic socket is needed. We predict that we will be able to foresee when a patient requires a new socket based on changes in their sock ply. With this knowledge, the information will be shared with practitioners with an expectation that it will allow them to change their daily practices to recognize the need for a new socket.

Vascular calcification (VC) is an indicator of atherosclerotic plaque burden and is a strong, independent predictor for future cardiovascular complications, significantly increasing the risk for cardiovascular morbidity and mortality. Type II diabetes (T2D), the 7th leading cause of death in the US, is associated with increased incidence of VC clinically, as well as in our recent animal study. Although VC was considered a degenerative process involving passive accumulation of calcium-phosphate salts associated with tissue necrosis, numerous studies have indicated that VC is an active, cell-mediated process that resembles embryonic bone formation and remodeling. Smooth muscle cells (SMCs) have been implicated in giving rise to bone- and cartilage-like cells in calcifying blood vessels by undergoing lineage reprogramming in response to local environmental cues. We investigated the association of S100A11, a calcium binding protein, with a multi-ligand receptor, the receptor for advanced glycation end products (RAGE), as a possible signaling mechanism involved in the lineage reprogramming of SMCs under T2D settings. We used a genetic fate mapping strategy to trace SM-derived cells in the model T2D mice that developed vascular calcification. Immunofluorescent dual staining for S100A11 and RAGE, along with immunohistochemical staining for Runx2/Cbfa1, an early osteochondrogenic marker, was performed on aortic sections of the T2D mice. The results revealed that S100A11-RAGE signaling is most likely to be involved in cartilage lesion development. This is supported by co-localization of S100A11-RAGE markers within cells of chondrocyte morphology in cartilaginous and calcific areas of blood vessels. Interestingly, almost all S100A11-RAGE positive cells were derived from SMCs that have been labeled by specific transgenes. These results highlight the significance of S100A11-RAGE signaling in VC and possibly provide for the development of preventative or therapeutic targets against T2D-related VC.

In recent years, increasing numbers of single amino acid mutations in cardiac troponin C (cTnC) have been identified in patients with cardiomyopathies. The troponin protein complex regulates cardiac muscle, as Ca2+ binding to cTnC initiates contraction. Previous studies of the mutations associated with hypertrophic cardiomyopathy (HCM) patients show increased Ca2+ sensitivity and of those with dilated cardiomyopathy (DCM) patients, decreased Ca2+ sensitivity. However, it is still unclear how a change in Ca2+ signaling might precipitate the disease, and unknown why a myofilament protein variant affects the Ca2+ transient, which is regulated by calcium handling proteins. This project focuses on the I61Q cTnC variant, which previous studies have shown decreases fraction shortening (produce weaker contractions) and Ca2+ binding of myofilaments. Determining how or if abundance and activity of sarcoplasmic reticulum (SR) proteins vary will shed light on the mechanism behind the change in Ca2+ transient. All cardiomyocytes were isolated from healthy adult rats and transfected by adenoviruses containing the I61Q gene. Transduced cardiomyocytes were cultured for 48 hours. Then, Western blotting determined abundance of Ca2+ handling proteins in the SR, such as phospholamban and SERCA-2.  SDS-Page gels were used to determine protein phosphorylation. Using imaging software to analyze the bands in the gel will result in a quantitative comparison of protein abundance and phosphorylation. The difference in protein expression and regulation provide insights for understanding the mechanism behind the change in Ca2+ sensitivity, and potentially allow I61Q to be delivered through gene therapy in patients with HCM to restore normal Ca2+ sensitivity. Past contractile measurements were performed at 25°C, but since the heart normally operates at 37°C, future experiments will be performed at this temperature for transduced cardiomyocytes. Contractions will be induced by electrical stimulation so that cell lengths, rates of sarcomere contraction/relaxation, and Ca2+ transients can be measured simultaneously using IonOptix video microscopy.

Cell transplantation has been shown to improve cardiac function following myocardial infarction (MI). The mechanisms behind improved function are not well-understood, but are correlated with changes in myofilament protein phosphorylation, which likely affect Ca2+- and length-dependent (preload) changes in force. This project was designed to test the hypotheses that 1) responsiveness to ventricular filling (Frank-Starling relationship) will decrease following MI, and 2) neonatal rat cardiomyocyte (NRC) transplantation after infarction will ameliorate this loss of function. One week after induction of MI, fluorescently-labeled NRCs were injected into the area of infarction of some of the animals. Sham-operated rats served as control. After 4-6 weeks, cardiac performance for all groups was preliminarily assessed using echocardiography. Then, whole working heart preparations were used measure cardiac power output, testing the heart’s ability to follow the Frank-Starling relationship. NRC-injected hearts exhibited higher average power and response to ventricular filling than sham (media-only) injected hearts, indicating that NRC-engraftment significantly ameliorates MI-induced decreases in preload responsiveness. Force and Ca2+ sensitivity (pCa50) of demembranated myocardial strips from these hearts were then assessed. NRC-injected hearts strips had a sarcomere length associated change in pCa50 that was similar to uninfarcted hearts (0.07 ± .02 vs. 0.10 ± .03, respectively), which was significantly improved as compared to strips from untreated, infarcted animals (0.0 ± .03). These results indicate that loss of preload dependence following MI is, at least in part, due to altered myofilament function and this is reversed following NRC engraftment. Current work involves investigating myofilament phosphorylation profiles to assess the mechanism of myocardial function changes. Preliminary results show decreased phosphorylation levels of cardiac troponin T (cTnT) in NRC-injected hearts compared to sham-operated and infarcted hearts. These changes in phosphorylation may be associated with the changes in force production following cell therapy.

Muscle contraction begins when Ca2+ binds to sTnC, which removes the inhibition of myosin binding to actin, allowing force development or shortening. Our laboratory has engineered sTnC variants with differential Ca2+ binding affinities to investigate how it affects healthy and diseased muscle. Chief among these variants are I60Q and M80Q, which have lower and higher Ca2+ binding affinities than the native protein, respectively. Expression of these variants in diseased or damaged muscle as a therapeutic treatment will be accomplished by viral transduction with cDNA. This method does not achieve 100% replacement of the native protein. Thus, my project is to determine the minimum variant level that needs to be replaced to have a signficant effect on contraction to help design treatment studies. Replacing native sTnC with I60Q or M80Q is also expected to gradually produce reduced or greater contraction when exposed to higher [Ca2+] prior to sTnC extraction and following reconstitution with variant:WT mixtures to determine force outputs. pCa50 is compared between pre-extraction, and reconstitution. Variants used in this study also contained polyhistidine tags, allowing us to quantify the amount of reconstitued protein incorporated into the fiber. These data show us the the effect of a particular reconstitution ratio and the amount of protein necessary to produce this effect. My preliminary data suggest that there is a progressive effect of increasing I60Q in muscle fibers to reduce the calcium sensitivity of force, thus reducing contractile strength. This variant may therefore be effective in reducing contraction in muscular dystrophy, thus maintaining muscle integrity and function.

 Myocardial infarction, heart failure, and congenitive heart disease are leading causes of morbidity and mortality worldwide. However, current cell therapies remain inadequate in addressing this clear medical need. One of the primary obstacles limiting the success of cell therapies is that stem cell derived cardiomyocytes (SCDCM’s) tend to be functionally immature, providing only a small fraction of the force generation of their matured counterparts. Thus, the work here focuses on developing a growth platform which incorporates topographical and mechanical properties in order to rapidly and thoroughly mature SCDCM’s. The field of tissue engineering is becoming increasingly aware of the roll that the extracellular matrix (ECM) plays in driving cellular differentiation and behavior, and the importance this has on the design of biomimetic scaffolds and growth platforms. A study of the native cardiac environment has revealed that the presence of aligned nanoscale matrix fibers plays a key role in establishing organization within the myocardium. This insight has allowed us to construct polymeric scaffolds which mimic the in vivo ECM, greatly improving cardiac culture techniques. There exists an optimal feature size and configuration at which nanogrooved topography is successful in promoting anisotropic alignment and elongation of cardiomyocytes, an increased action potential conduction velocity, and the substantial expression of cell-cell coupling proteins. Our current focus rests on understanding the way in which mechanical and chemical variations to the growth substrate can affect cell morphology and contractile properties when manipulated in conjunction with nanoscale topographical stimulation. This research will provide a strong foothold towards the development of a cardiac patch, which, implanted directly onto the infarct surface of the heart, could reduce scar tissue formation, promote healing and angiogenesis, and provide crucial contractile and mechanical strength.