The overall goal of this part of the laboratory’s program is to integrate specific signaling pathways with cardiac and cardiomyocyte function in the adult and during development, emphasizing the pathological implications of aberrant signaling and resultant stress on the cardiomyocyte. The laboratory uses the general approach of targeted mutagenesis along with cardiac-specific loss- and gain-of-function approaches, which can be modulated precisely. Each project is driven by specific hypotheses and experiments so that these methodologies are appropriate. Since the modifications can be controlled in a tissue and even cardiac-compartment-specific fashion, as well as at different developmental times, very precise genetic manipulations can be carried out effectively.
The cardiac-specific gain-of-function approach is particularly powerful if coupled with the use of inducible systems and a novel and robust, cardiac-specific inducible promoter, developed in the Robbins laboratory. A comprehensive analysis of the resultant mice can be a daunting task for a single lab, both in terms of time and expense, but the use of divisional cores greatly enhances the cost-effectiveness of the research. The Histopathology / Physiology Core provides a coherent centralized facility for generating cytological analyses where gross or subtle manifestations of the pathology or cytopathology resulting from the various targeting experiments can be discerned, integrated, and valuable correlations made between the different projects. Integration of these data directly with physiology aids in their integrative interpretation as well as pointing the way to the next experiments in order to extend the descriptive data into mechanism and function. The lab also has the capability of studying the entire functional spectrum of the modified cells, organ or whole animals, using a variety of invasive and noninvasive techniques, while another core provides a common source of cells suitable for transfection as well as the necessary adenovirus constructs and stocks.
A particular focus of the lab is the sarcomeric protein known as myosin binding protein C (MyBP-C). This is one of the most frequently mutated proteins found in human familial hypertrophic cardiomyopathy (FHC) and these alleles exhibit autosomal dominance. It is not clear as to whether the ensuing pathology is due to functional haploinsufficiency, the action(s) of a poison peptide or some combination. Complicating an understanding of the role or roles of truncated forms of cMyBP are our recent data showing the production of a stable 29kD fragment from cMyBP-C as a result of ischemia reperfusion injury and / or general cardiovascular stress. The fragment is stable but its functional capacity is unknown. Preliminary data indicate that it retains a capacity to influence contractile function, leading us to hypothesize that cMyBP-C fragments can play an important role in determining cardiomyocyte functionality.
Our objective is to explicitly define the functional and mechanistic outcomes of a series of cMyBP-C truncations that cause disease, and compare those data with the data obtained using the 29kD cMyBP-C fragment that is endogenously produced under stressed conditions. The central hypothesis is that while haploinsufficiency may play a role in the pathology of some of these mutations, a majority function as poison peptides, possibly through depression of thick-thin filament cycling. We are testing the hypothesis that the 29kD cMyBP-C fragment can function as a poison peptide by actively interfering with normal thick-thin filament interaction. Using a combination of isolated systems, in vitro cell culture and developmental stage-specific transgenesis, the function of this fragment will be determined. We are also testing the hypothesis that cMyBP-C truncations lead to pathology through haploinsufficiency or, more frequently, production of a poison peptide. A series of cMyBP-C’s carrying nonsense codons defined as causing human FHC will be made and used to replace endogenous cMyBP-C. The impact of the mutations on the cardiomyocyte’s general stress response and ensuing pathology will be determined and the pathogenic mechanism(s) determined.