Single-cell proteome heterogeneity due to MYH HCM mutations and recovery by Mavacamten

Abstract
Technological innovations continue to advance our understanding by delivering new insights and enabling
paradigm shifts that address previously unanswerable questions. Recent breakthroughs in high-sensitivity mass
spectrometry, coupled with innovative technological pipelines, have permitted our group, for the first time, to
directly quantify over 2,000 proteins from a single isolated cardiomyocyte (CM). This achievement provides a
precise proteomic profile of the heart’s contractile machinery.
Through single-cell proteomics (SCP), we have discovered that viable CMs isolated from healthy human and
mouse hearts form two to three distinct clusters characterized by significant differences in their sarcomeric and
mitochondrial proteomes. Additionally, CMs derived from mouse hearts harboring different hypertrophic
cardiomyopathy (HCM) mutations exhibit separate clustering, indicating profound heterogeneity in mitochondrial
and sarcomeric protein expression. Notably, this heterogeneity correlates not only with the specific mutation but
also with the mutant allele “dosage” within individual cells. These findings, replicated in iPSC-derived human
CMs (iCMs). These findings underscore our hypothesis that the mutation’s position along the myosin heavy
chain 7 (Myh7) gene influences not only the structure and function of the affected protein, which is also impacted
by the extent of “protein dosage” at the myofibrils and single CM. The resulting heterogeneity will impact the
broader sarcomeric organization, contractility, and metabolic function at the cellular level and ultimately the heart
architecture. Despite all mutations leading to HCM, their proteomic differences suggest they follow distinct
biochemical pathways. This implies that treatment of HCM patients should target these unique mechanisms. We
further hypothesize that each HCM-associated Myh7 mutation uniquely increases cardiomyocyte heterogeneity
at four levels: (i) protein structure and function; (ii) cellular proteomics; (iii) tissue localization within the
myocardium; and (iv) responses to the myosin inhibitor Mavacamten, which may affect individual dosing.
Our investigation will focus on three Myh7 mutations situated at different loci along the gene. Utilizing our
innovative proteomic analyses, informed by deep learning computational models, we aim to predict how these
biochemically distinct mutations affect subcellular structures, particularly at the myofibril level. These predictions
will be empirically tested, enabling us to refine our models. Additionally, we will employ two multiscale
approaches: mass spectrometry imaging of heart tissue and the expansion of our current multiscale filament
sarcomere model to encompass the entire ventricle. These approaches will help elucidate the architectural
differences among proteomic subgroups. Comprehensively understanding the effects of each mutation—at the
myofibrillar, cellular, and tissue levels—and their responses to Mavacamten will advance the development of
precision therapies for HCM. This will facilitate optimal dose determination and identify the need for additional,
yet-to-be-discovered interventions.