Tubulin Detyrosination: An Emerging Therapeutic Target in Hypertrophic Cardiomyopathy

Since the first reported series of patients with “cardiac hypertrophy of unknown cause” in 1944¹, insights into the primary pathogenesis and secondary consequences of hypertrophic cardiomyopathy (HCM) have advanced considerably. Beyond recognition that HCM is the most common genetic heart disease, there has been progress in elucidating the genetic origins of HCM, including a predominance of sarcomere protein mutations and typical autosomal dominant inheritance with variable penetrance. In a sizable subset of patients with HCM, there has been recognition that hypercontractility, due to increased myofilament calcium sensitivity, conspires with anatomic remodeling to produce dynamic left ventricular outflow tract obstruction (LVOTO). These insights inspired effective utilization of negative inotropic agents (beta-blockers, calcium-channel blockers and disopyramide) that often provide significant symptomatic relief in patients with LVOTO. When the anatomic features of HCM and LVOTO prove refractory to negative inotropes, alcohol septal ablation and surgical myectomy are highly effective at improving symptoms². Recently, more sophisticated targeting of the subcellular basis for hypercontractility has allowed development of direct myosin inhibitors that are highly effective at mitigating hypercontractility and LVOTO with a more favorable tolerability profile than previously employed agents. These recent successes, coupled with the ability to identify the specific pathogenic mutation in about half of patients with HCM, have inspired consideration of even more precise disease targeting, and perhaps even genotype-specific, therapeutics.

In this context, the manuscript by Schuldt et al³ in this issue of Circulation-Heart Failure reports the results of a proteomic analysis of myocardial tissue from a cohort of patients with HCM who had undergone septal myectomy for symptomatic LVOTO. The full cohort of patients with HCM was subdivided into 39 subjects who had an identified pathogenic sarcomere protein mutation and 11 in whom no pathogenic mutation was identified.

The proteomic profiling revealed that many of the abnormalities in myocardial expression of metabolic, extracellular matrix and muscle-related proteins in patients with HCM are similar among those with and without an identified sarcomere protein mutation. However, notable exceptions were abnormalities in cytoskeletal proteins, and particularly the abundance of detyrosinated α-tubulin, which was much greater among patients with HCM and a known sarcomere gene mutation than in those without an identified mutation. This particular finding is notable, because recent studies have shown that this specific post-translational modification - detyrosination of α-tubulin - has significant effects on the stability and density of the cardiomyocyte cytoskeleton and cell biomechanics. Specifically, increased detyrosination and associated changes to the cardiomyocyte microtubule network are causally linked to increased stiffness and viscoelasticity that reduce contractility and slow both contraction and relaxation^4-8.

To better define the functional significance of increased tubulin detyrosination in “genotype-positive” patients, the Schuldt et al performed studies using a murine model designed to mimic the most severely affected genotype in the HCM patient cohort: MYBPC3_2373insG. In these studies, mice homozygous for this mutation exhibited particularly severe hypertrophy, a reduced ejection fraction and severe relaxation abnormalities. Moreover, hearts from mice with this analogue of the human mutation had markedly increased levels of detyrosinated tubulin compared with wild type controls, and cardiomyocytes from these mice demonstrated slowed contraction and relaxation. Importantly, administration of parthenolide, an agent known to reduce the proportion of detyrosinated tubulin, normalized the cardiomyocyte contraction and relaxation times, suggesting that increased detyrosination contributes to the contractility and relaxation defects in mice (and humans) carrying the MYBPC3_2373insG mutation. Compared with a second mouse strain with a different Mybpc3 mutation, less severe increases in tubulin detyrosination and slower progression of hypertrophy and contractile defects, the mice carrying the MYBPC3_2373insG mutation developed severe remodeling at a much earlier age. Though parthenolide has actions that are independent of decreased detyrosination⁹, the authors conclude that increased detyrosination of microtubules contributes to cardiomyocyte stiffness and dysfunction with a greater impact in the presence of a pathogenic sarcomere protein mutation.

To a large extent, the findings by Schuldt et al confirm and complement previous reports demonstrating increased tubulin detyrosination in hearts obtained from patients with advanced HCM requiring heart transplantation^4,5. In those studies, parthenolide or genetic manipulation of the enzymes of the detyrsosination/tyrosination cycle improved contractility and relaxation velocities in intact human cardiomyocytes isolated from patients with HCM^5,6. In this context, the studies by Schuldt et al using myectomy samples suggest that the development of functionally significant tubulin detyrosination in HCM may occur long before end-stage heart disease requiring transplantation.

The interpretation of the findings in the smaller subset of HCM patients without a pathogenic mutation, who exhibited more modest increases in tubulin detyrosination, is less clear. While Schuldt et al conclude that the presence or absence of a demonstrable sarcomeric mutation determines the pathogenic contribution of tubulin detyrosination, the significant differences in the absolute degree of hypertrophy in the HCM groups with and without sarcomere protein mutations represents a potential confounding factor because the patients without a sarcomere protein mutation had significantly less hypertrophy than those with an identified mutation. Specifically, among patients without an identified mutation none had a septal thickness over 20 mm. In contrast, 19 of 39 patients with a pathogenic mutation had a septal thickness greater than 20 millimeters; and 17 of 18 patients of those with a MYBPC3 mutation had a septal thickness ≥ 20 mm. Thus, it could be the degree of pathologic hypertrophy that drives the magnitude of a-tubulin detyrosination among patients with HCM, irrespective of mutation status. Indeed, the aforementioned studies using hearts from transplant recipients^4,5, demonstrate that patients with advanced dilated cardiomyopathy also demonstrated substantially increased degrees of tubulin detyrosination, which was not observed in nonfailing hearts with compensated hypertrophy. The temporal progression of this post-translational modification and its correlative or causative link to the initiation, establishment and progression of hypertrophy requires further inquiry in controlled research models.

The imperfect modeling of human HCM with mouse models is another shortcoming worth noting. In contrast with the heterozygous MYBPC3 mutations in the patients classified as genotype positive by Schuldt et al, the murine models employed for their longitudinal and isolated myocyte studies are homozygous. This is an important difference that likely exacerbates and accelerates the progression of HCM in mice, and could affect the magnitude of downstream changes including tubulin detyrosination.

Nevertheless, the current studies further advance the concept that the myocardial adaptations downstream of primary genetic defects may be opportunities for therapeutic targeting. In particular, microtubule-dependent cardiomyocyte stiffening, as a consequence of reversible, post-transcriptional detyrosination of a-tubulin, is a promising therapeutic target that is supported by both the human proteomics and mouse studies in the manuscript by Schuldt et al. In addition to clearer elucidation of the degree to which specific pathogenic mutations drive tubulin detyrosination, independent of hypertrophy magnitude, further studies should examine whether in vivo targeting of tubulin detyrosination improves contractile and relaxation defects at the organ level and/or alters the progression of HCM and its associated morbidity.