The Role of Pointed End Binding Proteins in Cardiac Thin Filament Actin Regulation and the Development of Dilated Cardiomyopathy

Abstract

Dilated cardiomyopathy (DCM) is a disease of the heart muscle that is characterized by thinning and enlargement of one or both pumping chambers of the heart (the ventricles). Clinically, DCM is associated with a high rate of morbidity and mortality due to the inefficient pumping of the heart and presence of life-threatening arrhythmias. Mutations in a diverse group of genes result in DCM, with genetic mutations now being identified in up to 40-50% of all DCM patients. Due to this high mutagenic rate a much better understanding of the genetic pathologies associated with DCM is needed. Recently, the first human mutations in two sarcomeric proteins, Lmod2 and CAP2, were discovered in pediatric patients who presented with early onset DCM. Exome sequencing revealed a bi-allelic nonsense mutation in the LMOD2 gene (c.1193G>A) and a recessive deleterious mutation in CAP2 (c.636+1G>A). It has previously been demonstrated that leiomodin2 (Lmod2) is a sarcomeric protein known to tightly regulate actin-thin filament lengths by acting as a nucleator of actin polymerization, resulting in thin filament elongation. Likewise, it has been demonstrated that cyclase-associated protein 2 (CAP2) depolymerizes filamentous actin (F-actin) and plays a role in cardiomyocyte maturation. Together with the sarcomeric protein tropomodulin (Tmod), which serves to shorten thin filaments, Lmod2 is able to precisely fine-tune thin filament lengths in the sarcomere while CAP2 is able to stabilize nascent myofibrils. Thus, all three proteins contribute to the overall structure and contractile properties of cardiac muscle. Based on Lmod2’s and CAP2’s function in the heart we hypothesize that mutations in these sarcomeric proteins result in severe cardiac dysfunction and induce cardiomyopathy due to altered actin regulation. To better understand the consequences of these mutations and the role of these proteins at the level of the sarcomere we have: (1) generated patient iPSC-derived cardiomyocytes (hiPSC-CMs) harboring the LMOD2 mutation and a gene-corrected CRISPR/Cas9 isogenic control line, and (2) used neonatal rat ventricular cardiomyocytes along with biochemical assays to study Lmod2 and CAP2 function, respectively. Using human iPSC-CMs we discovered structural changes in Lmod2 mutant cardiomyocytes at the level of the sarcomere, shortened actin-thin filament lengths, altered contractile properties and compromised ability to regulate calcium flux. Furthermore, significant differences in the transcriptome network and differentially expressed genes involved in sarcomere function and cardiomyocyte maturation were discovered between mutant and isogenic controls. A key pathological pathway, the serum response factor (SRF) pathway, involved in cell growth, proliferation and muscle development was discovered to be downregulated in hiPSC-CMs. In parallel, the in vivo effects of the LMOD2 mutation were examined through the use of a CRISPR/Cas9 mouse model harboring the homologous mutation to the patient. Transcriptome analysis of mutant mice found significantly altered contractile and calcium regulatory pathways, compared to wildtype mice. Therefore, the goal of these studies was to effectively translate a patient’s clinical genotype into human iPSC-CMs while simultaneously examining the disease phenotype through the use of a novel rodent model. Together, these experiments provide important information on Lmod2, an understudied sarcomeric protein, that is necessary for normal heart function. Simultaneously, we discovered that CAP2 has essential functions in regulating actin dynamics at the pointed end of cardiac thin filaments and that CAP2 plays an important role in myofibril maturation. We further determined that CAP2 may be influencing the regulation of Lmod2 and Tmod1 actin dynamics and proposed a model of how all three pointed-end binding proteins work together. The findings from these studies highlight the physiological relevance of these proteins in the sarcomere and provide further insight into the functions of a novel protein, CAP2, within the context of two well-studied actin regulators, Lmod2 and Tmod1. By deciphering the CAP2 interactome and its role in the cardiac sarcomere, a better understanding of the severe disease pathology that results from CAP2 mutations, can be achieved.