Author + information
- Lili Wang, PhD,
- Dmytro O. Kryshtal, PhD,
- Kyungsoo Kim, PhD,
- Shan Parikh, MS,
- Adrian Gabriel Cadar, PhD,
- Kevin Richard Bersell, PhD,
- Huan He, PhD,
- Jose R. Pinto, PhD and
- Bjorn C. Knollmann, MD, PhD∗ ()
- ↵∗Vanderbilt Center for Arrhythmia Research and Therapeutics (VanCART), Division of Clinical Pharmacology, Vanderbilt University Medical Center, Medical Research Building IV, Room 1265, 2215B Garland Avenue, Nashville, Tennessee 37232-0575
Familial hypertrophic cardiomyopathy is caused by mutations in genes encoding sarcomere proteins. Among hypertrophic cardiomyopathy–linked disease genes, cardiac troponin T (TnT) mutations are associated with a high incidence of arrhythmic cardiac death (1), but the underlying mechanism has remained elusive. Studies in mice suggest that increased myofilament Calcium (Ca) buffering caused by pathogenic TnT mutations such as TnT-I79N generates susceptibility to ventricular arrhythmias by Ca-dependent action potential (AP) remodeling (2). As cardiac electrophysiological properties of mice differ substantially from humans, here we used cardiomyocytes (CMs) derived from human-induced pluripotent stem cells (hiPSCs) to study the effect of increasing myofilament Ca sensitivity on cytosolic Ca buffering and cardiac AP.
We first generated hiPSC lines from dermal fibroblasts of 3 healthy donors using standard approaches. One of the 3 hiPSC lines was edited using CRISPR/Cas9 and a heterozygous Ca-sensitizing TnT-I79N mutation introduced. Hence, the unedited line was the isogenic control line. The other 2 lines were named population controls. We then used the Matrigel mattress method (3) to generate single rod-shaped CMs for each iPSC line and studied TnT-I79N protein levels, cytosolic Ca buffering, and electrophysiology.
Analysis by nano-liquid chromatography mass spectrometry indicated that 43% of total TnT protein was mutant in I79N hiPSC-CMs. Cytosolic Ca buffering was quantified in voltage-clamped hiPSC-CMs as reported in Hwang et al. (4). Cytosolic Ca binding affinity was significantly higher (= lower Kd values) in I79N compared to both isogenic control and population control hiPSC-CMs (Figure 1A). Furthermore, application of the Ca sensitizer EMD57033 (EMD, 3 μmol/l), which increases Ca sensitivity comparable to I79N (2), significantly lowered Kd values of population control hiPSC-CMs (Figure 1A). Maximal binding capacity Bmax, an estimate of total cytoplasmic Ca binding sites, was not altered by I79N or EMD treatment (data not shown).
We then asked if increasing myofilament Ca buffering alters the human AP. APs were recorded in current clamp mode without exogenous Ca buffers added to intracellular solutions and analyzed as described in Hondeghem et al. (5) during steady-state pacing at 0.5 Hz. Resting membrane potential, AP amplitude, and AP upstroke velocity were comparable in all groups (data not shown). Compared to isogenic controls, APs of I79N hiPSC-CM were more triangulated (i.e., shorter early repolarization [action potential duration 50%, APD50] without changes in late repolarization [APD90]) (Figure 1B) and exhibited beat-to-beat AP instability (average ratio of interquartile range to median APD50: I79N 0.12 ± 0.02 vs. isogenic control 0.07 ± 0.01; n = 17 to 21, p < 0.05). Increasing buffering with 3 μmol/l EMD had the same triangulating effect in isogenic and population control hiPSC-CMs (Figure 1B). Afterdepolarizations were not observed. Pre-treatment with the Ca desensitizer blebbistatin (3 μmol/l) or excess cytosolic buffering (by adding 14 mmol/l ethylene glycol tetraacetic acid to intracellular solutions) prevented AP triangulation (Figure 1B). These results provide strong evidence that increased myofilament Ca buffering causes AP triangulation and AP instability.
How could increased myofilament Ca buffering alter the human cardiac AP? Any increased Ca buffering should reduce intracellular free [Ca]. We found that I79N hiPSC-CMs had lower peak Ca transients (data not shown) analogous to results obtained in I79N mice (2). As cellular Ca removal by the sodium calcium exchanger (NCX) generates inward current, lowering [Ca] would decrease inward NCX current and therefore shorten the AP, especially during early repolarization when intracellular [Ca] reaches its peak. To test this hypothesis, NCX-mediated Ca extrusion was blocked by rapidly replacing extracellular sodium (Na+) with lithium (Li+). NCX block with Li+ eliminated the AP differences between I79N and isogenic control hiPSC-CMs (Figure 1B).
Taken together, our results indicate that myofilament-dependent increase in cytosolic Ca buffering shortens early repolarization via a Ca-dependent, NCX-mediated mechanism, resulting in AP triangulation and AP instability, both of which are well-established predictors of increased arrhythmia risk (5). Our results not only provide novel insight into the regulation of the human cardiac AP by myofilament Ca buffering, but also suggest a new mechanism of how a pathogenic TnT mutation causes pro-arrhythmic AP changes in human CMs.
Please note: Supported in part by grants from the U.S. National Institutes of Health (R01HL71670, R01HL128044, R01HL124935 to BCK, R01 HL128683 to JRP). Mr. Parikh was supported by NIGMS T32 GM07347 through the Vanderbilt Medical-Scientist Training Program and NHLBI F30 HL131179. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. Wang and Kryshtal contributed equally to this work.
- 2017 American College of Cardiology Foundation
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