The Shaw Laboratory is focused on basic myocardial biology. We define the mechanisms by which the cytoskeleton delivers ion channels to their respective subregions on the cardiac membrane, and how delivery is changed during heart failure. Our work defined the paradigm of Targeted Delivery which describes how the cytoskeleton delivers membrane proteins directly to their functional membrane subdomain and why there is less delivery in failing heart. Our models involve exploration of Connexin43 gap junction trafficking to the cardiomyocyte intercalated disc and L-type calcium channel trafficking to cardiomyocyte T-tubules. In the process we identified that the mRNA of Cx43 is alternatively translated to generate endogenously up to six different truncated isoforms which are essential to trafficking. These isoforms we identified have a different biophysics and biology as Connexin43 and thus function as new proteins with important roles in basic cell biology. The most commonly translated isoform, GJA1-20k, is critical for Connexin43 forward trafficking as well as mitochondrial organization, transport and function. A major current focus of my group is to identify how GJA1-20k works with the actin cytoskeleton to regulate both membrane protein trafficking and metabolism. A second major focus involves another trafficking molecule Dr. TingTing Hong and I cloned, cardiac BIN1 which regulates L-type calcium channel trafficking, T-tubule folds and is also released into blood as T-tubule origin microparticles, and available as a biomarker of muscle remodeling.
- Targeted Delivery. We asked the question how do the newly made and short-lived gap junction proteins arrive at the intercalated disc? The result of these mechanistic explorations is the Targeted Delivery model Cx43 trafficking but which newly formed hemichannels exit the Golgi and ride microtubule highways specifically to their destination membrane subdomains. Specificity of delivery is a combination of the channel protein, microtubule plus-end tracking proteins, and a membrane anchor. We have found Targeted Delivery occurs for Cx43 trafficking in cardiomyocytes, and is disrupted in conditions of oxidative stress, helping to explain decreased cell-cell coupling in failing myocardium, and that actin organized the microtubule highways by the alternatively translated GJA1-20k isoform. Targeted Delivery also works for L-type calcium channel delivery to T-tubules and has become the prevailing model for channel trafficking in non-failing hearts and explains channel mislocalization in heart failure.
2. GJA1-20k in Trafficking and Metabolism. A byproduct of our Cx43 trafficking studies revealed that Cx43 mRNA which is the product of a single coding exon (hence no splicing), can generate not one but seven total proteins with the latter six encoded with internal AUG start sites and translated by Alternative Translation. Furthermore, translation of the smaller isoforms is mTOR dependent and the 20kd smaller isoform (GJA1-20k) is important for Cx43 forward trafficking. Further exploring the roles of the smaller isoforms, we are finding that actin is important to Cx43 forward delivery, and that mitochondria are affected by GJA1-20k as well with function that include mitochondrial protection and mediating ischemic preconditioning, as well as mediating mitochondrial biogenesis and mitochondrial fission:
3. Biomarker and Therapeutic Development for Heart Failure. A translational component to our effort uses the lessons learned from mechanistic studies of cardiomyocyte trafficking to develop novel diagnostics and therapeutics. The most advanced of such studies is use of blood based cBIN1 as an index of cardiac health. We have found that not only is cBIN1 important to calcium hemostasis and electrical stability in ventricular cells, but is reduced in acquired heart failure and is turned over into blood in levels that reflect cardiac content. We have helped developed a cBIN1 score (CS) as a first of its kind biomarker of cardiac muscle health that could be used as a screen for heart failure, prognosticate heart failure outcomes, and prognosticate the occurrence of ventricular arrhythmia. CS is now available by Sarcotein Diagnostics for research use (www.sarcotein.com).