Myocardial Recovery With Mechanical Circulatory Support Is Linked to Alternative Splicing and Subcellular Localization of CAMK2D

Abstract

BACKGROUND: Cardiac reverse remodeling occurs in a small subset of patients with heart failure treated with guideline-directed therapies. This phenomenon, which is defined by reduced ventricular dilatation and improved systolic function, is most common in patients receiving left ventricular assist device (LVAD) therapy. Identifying therapeutic targets for initiating reverse remodeling is an area of great clinical interest, because these patients experience improved outcomes and quality of life. Targets may be discovered among the unique molecular changes associated with LVAD-induced partial myocardial functional recovery; however, the mechanisms underlying this favorable response are incompletely understood. METHODS: To identify molecular signatures of recovery, we studied paired pre-LVAD and post-LVAD myocardial samples from patients with heart failure who received LVAD as a bridge to transplant (10 responders and 9 nonresponders) and controls without heart failure. We performed bulk RNA sequencing, tandem mass tag quantitative proteomics, and tandem mass tag quantitative phosphoproteomics with follow-up mechanistic and functional investigations in primary rat cardiomyocytes and human engineered heart tissues. RESULTS: Alternative RNA splicing was the leading pathway associated with a favorable response to LVAD. Responders had increased RNA splicing factor expression and unique gene splice variant expression compared with nonresponders. Alternative splicing of CAMK2D (calcium/calmodulin-dependent protein kinase 2δ) was a particularly strong predictor of recovery; increased inclusion of exon 14, which encodes the nuclear splice variant CAMK2D-B, was inversely correlated with functional recovery. Nonresponders also displayed hyperphosphorylation near the nuclear localization signal in CAMK2D-B. Investigations in primary cardiomyocytes and subcellular organelle fractions from the human hearts revealed that nuclear localization signal phosphorylation prevented adrenergic stress-dependent nuclear targeting of CAMK2D-B. Expression of a cytoplasm-restricted CAMK2D-B uniquely remodeled the phosphoproteome of primary rat cardiomyocytes compared with a nuclear-competent version and blunted calcium transients in engineered heart tissues. CONCLUSIONS: This is the first study to integrate transcriptome, alternative transcriptome, proteome, and phosphoproteome analyses of heart samples from LVAD-supported patients to investigate myocardial recovery. We identified that increased expression and phosphorylation of the nuclear CAMK2D splice variant predicted poor outcomes. This phosphorylation restricted CAMK2D-B to the cytosol, leading to impaired cardiomyocyte calcium handling. These findings suggest that LVAD nonresponder patients may benefit from therapies that modulate subcellular localization of CAMK2D or inhibit its activity.