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Published on: January 16, 2019
A Precision Medicine Approach to the Rescue of Function on Malignant Calmodulinopathic Long-QT Syndrome
Worawan B Limpitikul1, Ivy E Dick1, David J Tester1
1From the Calcium Signals Laboratory, Department of Biomedical Engineering (W.B.L., I.E.D., W.Y., M.H.C., J.B., D.T.Y.) and Division of Cardiology, Department of Medicine (P.L., D.D., G.F.T.), The Johns Hopkins University School of Medicine, Baltimore, MD; Department of Physiology, The University of Maryland School of Medicine, Baltimore (I.E.D.); Division of Heart Rhythm Services, Department of Cardiovascular Diseases (D.J.T., N.J.B., M.J.A.), Division of Pediatric Cardiology, Department of Pediatrics (D.J.T., N.J.B., M.J.A.), and Windland Smith Rice Sudden Death Genomics Laboratory, Department of Molecular Pharmacology and Experimental Therapeutics (D.J.T., N.J.B., M.J.A.), Mayo Clinic, Rochester, MN; and Division of Cardiology, Nicklaus Children's Hospital, Miami, FL (R.J.K.).
Rationale:
Calmodulinopathies comprise a new category of potentially life-threatening genetic arrhythmia syndromes capable of producing severe long-QT syndrome (LQTS) with mutations involving CALM1, CALM2, or CALM3. The underlying basis of this form of LQTS is a disruption of Ca2+/calmodulin (CaM)-dependent inactivation of L-type Ca2+ channels.
Objective:
To gain insight into the mechanistic underpinnings of calmodulinopathies and devise new therapeutic strategies for the treatment of this form of LQTS.
Methods And Results:
We generated and characterized the functional properties of induced pluripotent stem cell-derived cardiomyocytes from a patient with D130G-CALM2-mediated LQTS, thus creating a platform with which to devise and test novel therapeutic strategies. The patient-derived induced pluripotent stem cell-derived cardiomyocytes display (1) significantly prolonged action potentials, (2) disrupted Ca2+ cycling properties, and (3) diminished Ca2+/CaM-dependent inactivation of L-type Ca2+ channels. Next, taking advantage of the fact that calmodulinopathy patients harbor a mutation in only 1 of 6 redundant CaM-encoding alleles, we devised a strategy using CRISPR interference to selectively suppress the mutant gene while sparing the wild-type counterparts. Indeed, suppression of CALM2 expression produced a functional rescue in induced pluripotent stem cell-derived cardiomyocytes with D130G-CALM2, as shown by the normalization of action potential duration and Ca2+/CaM-dependent inactivation after treatment. Moreover, CRISPR interference can be designed to achieve selective knockdown of any of the 3 CALM genes, making it a generalizable therapeutic strategy for any calmodulinopathy.
Conclusions:
Overall, this therapeutic strategy holds great promise for calmodulinopathy patients as it represents a generalizable intervention capable of specifically altering CaM expression and potentially attenuating LQTS-triggered cardiac events, thus initiating a path toward precision medicine.
Insights
Calmodulinopathies cause severe long-QT syndrome (LQTS) by disrupting calcium channel function. Gene suppression using CRISPR interference offers a promising therapeutic strategy to restore normal heart function in affected patients.
Area of Science:
- Cardiovascular Genetics
- Molecular Cardiology
- Genetic Medicine
Background:
- Calmodulinopathies are a class of genetic arrhythmia syndromes linked to mutations in CALM1, CALM2, or CALM3 genes.
- These mutations disrupt Ca2+/calmodulin (CaM)-dependent inactivation of L-type Ca2+ channels, leading to severe long-QT syndrome (LQTS).
Purpose of the Study:
- To investigate the mechanistic basis of calmodulinopathies.
- To develop novel therapeutic strategies for LQTS caused by calmodulin mutations.
Main Methods:
- Generated patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with a D130G-CALM2 mutation.
- Utilized CRISPR interference (CRISPRi) to selectively suppress the mutant CALM2 gene in iPSC-CMs.
- Assessed functional rescue by measuring action potential duration and Ca2+/CaM-dependent inactivation.
Main Results:
- Patient-derived iPSC-CMs exhibited prolonged action potentials and disrupted Ca2+ cycling.
- CRISPRi-mediated suppression of CALM2 normalized action potential duration and Ca2+/CaM-dependent inactivation.
- The CRISPRi strategy is generalizable to all three CALM genes, applicable to any calmodulinopathy.
Conclusions:
- CRISPR interference presents a viable and generalizable therapeutic approach for calmodulinopathies.
- This strategy can specifically modulate CaM expression to mitigate LQTS-related cardiac events.
- This work paves the way for precision medicine interventions in genetic arrhythmia syndromes.
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