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In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes
Published on: June 22, 2020
Depletion of Vasohibin 1 Speeds Contraction and Relaxation in Failing Human Cardiomyocytes
Christina Yingxian Chen1, Alexander K Salomon1, Matthew A Caporizzo1
1From the Department of Physiology, Pennsylvania Muscle Institute (C.Y.C., A.K.S., M.A.C., S.C., N.A.K., A.I.B., K.B.M., B.L.P.), University of Pennsylvania Perelman School of Medicine, Philadelphia.
Insights
In heart failure, detyrosinated microtubules stiffen heart cells. Inhibiting VASH1-SVBP complexes or activating TTL enzymes can reduce stiffness and improve relaxation in failing cardiomyocytes.
Area of Science:
- Cardiovascular Biology
- Cellular Mechanics
- Molecular Cardiology
Background:
- Impaired myocardial relaxation is a key feature of heart failure (HF).
- Detyrosinated microtubules contribute to cardiomyocyte stiffening and impaired relaxation in human HF.
- The enzymes responsible for detyrosination have remained unclear, limiting therapeutic development.
Purpose of the Study:
- To investigate if the VASH1/2-SVBP complex acts as an active detyrosinase in cardiomyocytes.
- To determine if inhibiting VASH-SVBP can reduce stiffness and improve contractility in HF.
Main Methods:
- Transcriptional profiling to assess VASH1 and VASH2 abundance in human hearts.
- Short hairpin RNA (shRNA) mediated knockdown of VASH1, VASH2, and SVBP.
- Enzymatic assays using a catalytically dead TTL mutant (TTL-E331Q) to differentiate detyrosination from microtubule depolymerization.
- Assessment of cardiomyocyte contractility and calcium transients.
- Atomic force microscopy to measure cardiomyocyte stiffness.
Main Results:
- VASH1 transcript is significantly more abundant than VASH2 in human hearts.
- VASH1-SVBP and VASH2-SVBP complexes function as tubulin carboxypeptidases, with VASH1 playing a dominant role.
- VASH1 depletion improved contractile kinetics in failing cardiomyocytes and reduced stiffness.
- TTL, but not TTL-E331Q, accelerated relaxation, indicating enzymatic activity is crucial.
- VASH1 depletion improved kinetics independently of calcium cycling alterations.
Conclusions:
- VASH-SVBP complexes are active tubulin carboxypeptidases in cardiomyocytes.
- Inhibiting VASH1 or activating TTL can decrease cardiomyocyte stiffness and enhance relaxation in HF.
- Targeting detyrosination presents a potential therapeutic strategy for diastolic dysfunction in heart failure.
Rationale:
Impaired myocardial relaxation is an intractable feature of several heart failure (HF) causes. In human HF, detyrosinated microtubules stiffen cardiomyocytes and impair relaxation. Yet the identity of detyrosinating enzymes have remained ambiguous, hindering mechanistic study and therapeutic development.
Objective:
We aimed to determine if the recently identified complex of VASH1/2 (vasohibin 1/2) and SVBP (small vasohibin binding protein) is an active detyrosinase in cardiomyocytes and if genetic inhibition of VASH-SVBP is sufficient to lower stiffness and improve contractility in HF.
Methods And Results:
Transcriptional profiling revealed that VASH1 transcript is >10-fold more abundant than VASH2 in human hearts. Using short hairpin RNAs (shRNAs) against VASH1, VASH2, and SVBP, we showed that both VASH1- and VASH2-SVBP complexes function as tubulin carboxypeptidases in cardiomyocytes, with a predominant role for VASH1. We also generated a catalytically dead version of the tyrosinating enzyme TTL (TTL-E331Q) to separate the microtubule depolymerizing effects of TTL from its enzymatic activity. Assays of microtubule stability revealed that both TTL and TTL-E331Q depolymerize microtubules, while VASH1 and SVBP depletion reduce detyrosination independent of depolymerization. We next probed effects on human cardiomyocyte contractility. Contractile kinetics were slowed in HF, with dramatically slowed relaxation in cardiomyocytes from patients with HF with preserved ejection fraction. Knockdown of VASH1 conferred subtle kinetic improvements in nonfailing cardiomyocytes, while markedly improving kinetics in failing cardiomyocytes. Further, TTL, but not TTL-E331Q, robustly sped relaxation. Simultaneous measurements of calcium transients and contractility demonstrated that VASH1 depletion speeds kinetics independent from alterations to calcium cycling. Finally, atomic force microscopy confirmed that VASH1 depletion reduces the stiffness of failing human cardiomyocytes.
Conclusions:
VASH-SVBP complexes are active tubulin carboxypeptidases in cardiomyocytes. Inhibition of VASH1 or activation of TTL is sufficient to lower stiffness and speed relaxation in cardiomyocytes from patients with HF, supporting further pursuit of detyrosination as a therapeutic target for diastolic dysfunction.
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