Dataset Information

Myofilament Ca sensitization increases cytosolic Ca binding affinity, alters intracellular Ca homeostasis, and causes pause-dependent Ca-triggered arrhythmia.

ABSTRACT:

Rationale

Ca binding to the troponin complex represents a major portion of cytosolic Ca buffering. Troponin mutations that increase myofilament Ca sensitivity are associated with familial hypertrophic cardiomyopathy and confer a high risk for sudden death. In mice, Ca sensitization causes ventricular arrhythmias, but the underlying mechanisms remain unclear.

Objective

To test the hypothesis that myofilament Ca sensitization increases cytosolic Ca buffering and to determine the resulting arrhythmogenic changes in Ca homeostasis in the intact mouse heart.

Methods and results

Using cardiomyocytes isolated from mice expressing troponin T (TnT) mutants (TnT-I79N, TnT-F110I, TnT-R278C), we found that increasing myofilament Ca sensitivity produced a proportional increase in cytosolic Ca binding. The underlying cause was an increase in the cytosolic Ca binding affinity, whereas maximal Ca binding capacity was unchanged. The effect was sufficiently large to alter Ca handling in intact mouse hearts at physiological heart rates, resulting in increased end-diastolic [Ca] at fast pacing rates, and enhanced sarcoplasmic reticulum Ca content and release after pauses. Accordingly, action potential (AP) regulation was altered, with postpause action potential prolongation, afterdepolarizations, and triggered activity. Acute Ca sensitization with EMD 57033 mimicked the effects of Ca-sensitizing TnT mutants and produced pause-dependent ventricular ectopy and sustained ventricular tachycardia after acute myocardial infarction.

Conclusions

Myofilament Ca sensitization increases cytosolic Ca binding affinity. A major proarrhythmic consequence is a pause-dependent potentiation of Ca release, action potential prolongation, and triggered activity. Increased cytosolic Ca binding represents a novel mechanism of pause-dependent arrhythmia that may be relevant for inherited and acquired cardiomyopathies.

SUBMITTER: Schober T

PROVIDER: S-EPMC3393041 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Publications

Myofilament Ca sensitization increases cytosolic Ca binding affinity, alters intracellular Ca homeostasis, and causes pause-dependent Ca-triggered arrhythmia.

Schober Tilmann T Huke Sabine S Venkataraman Raghav R Gryshchenko Oleksiy O Kryshtal Dmytro D Hwang Hyun Seok HS Baudenbacher Franz J FJ Knollmann Björn C BC

Circulation research 20120529 2

<h4>Rationale</h4>Ca binding to the troponin complex represents a major portion of cytosolic Ca buffering. Troponin mutations that increase myofilament Ca sensitivity are associated with familial hypertrophic cardiomyopathy and confer a high risk for sudden death. In mice, Ca sensitization causes ventricular arrhythmias, but the underlying mechanisms remain unclear.<h4>Objective</h4>To test the hypothesis that myofilament Ca sensitization increases cytosolic Ca buffering and to determine the res ...[more]

PMID: 22647877

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Project description:Bradycardia prolongs action potential (AP) durations (APD adaptation), enhances dispersion of repolarization (DOR), and promotes tachyarrhythmias. Yet, the mechanisms responsible for enhanced DOR and tachyarrhythmias remain largely unexplored. Ca(2+) transients and APs were measured optically from Langendorff rabbit hearts at high (150 × 150 ?m(2)) or low (1.5 × 1.5 cm(2)) magnification while pacing at a physiological (120 beats/min) or a slow heart rate (SHR = 50 beats/min). Western blots and pharmacological interventions were used to elucidate the regional effects of bradycardia. As a result, bradycardia (SHR 50 beats/min) increased APDs gradually (time constant ?f?s = 48 ± 9.2 s) and caused a secondary Ca(2+) release (SCR) from the sarcoplasmic reticulum during AP plateaus, occurring at the base on average of 184.4 ± 9.7 ms after the Ca(2+) transient upstroke. In subcellular imaging, SCRs were temporally synchronous and spatially homogeneous within myocytes. In diastole, SHR elicited variable asynchronous sarcoplasmic reticulum Ca(2+) release events leading to subcellular Ca(2+) waves, detectable only at high magnification. SCR was regionally heterogeneous, correlated with APD prolongation (P < 0.01, n = 5), enhanced DOR (r = 0.9277 ± 0.03, n = 7), and was gradually reversed by pacing at 120 beats/min along with APD shortening (P < 0.05, n = 5). A stabilizer of leaky ryanodine receptors (RyR2), 3-(4-benzylcyclohexyl)-1-(7-methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)propan-1-one (K201; 1 ?M), suppressed SCR and reduced APD at the base, thereby reducing DOR (P < 0.02, n = 5). Ventricular ectopy induced by bradycardia (n = 5/15) was suppressed by K201. Western blot analysis revealed spatial differences of voltage-gated L-type Ca(2+) channel protein (Cav1.2?), Na(+)-Ca(2+) exchange (NCX1), voltage-gated Na(+) channel (Nav1.5), and rabbit ether-a-go-go-related (rERG) protein [but not RyR2 or sarcoplasmic reticulum Ca(2+) ATPase 2a] that correlate with the SCR distribution and explain the molecular basis for SCR heterogeneities. In conclusion, acute bradycardia elicits synchronized subcellular SCRs of sufficient magnitude to overcome the source-sink mismatch and to promote afterdepolarizations.

Project description:Mutations in thin filament regulatory proteins that cause hypertrophic cardiomyopathy (HCM) increase myofilament Ca2+ sensitivity. Mouse models exhibit increased Ca2+ buffering and arrhythmias, and we hypothesized that these changes are primary effects of the mutations (independent of compensatory changes) and that increased Ca2+ buffering and altered Ca2+ handling contribute to HCM pathogenesis via activation of Ca2+-dependent signaling. Here, we determined the primary effects of HCM mutations on intracellular Ca2+ handling and Ca2+-dependent signaling in a model system possessing Ca2+-handling mechanisms and contractile protein isoforms closely mirroring the human environment in the absence of potentially confounding remodeling. Using adenovirus, we expressed HCM-causing variants of human troponin-T, troponin-I, and α-tropomyosin (R92Q, R145G, and D175N, respectively) in isolated guinea pig left ventricular cardiomyocytes. After 48 h, each variant had localized to the I-band and comprised ∼50% of the total protein. HCM mutations significantly lowered the Kd of Ca2+ binding, resulting in higher Ca2+ buffering of mutant cardiomyocytes. We observed increased diastolic [Ca2+] and slowed Ca2+ reuptake, coupled with a significant decrease in basal sarcomere length and slowed relaxation. HCM mutant cells had higher sodium/calcium exchanger activity, sarcoplasmic reticulum Ca2+ load, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) activity driven by Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of phospholamban. The ryanodine receptor (RyR) leak/load relationship was also increased, driven by CaMKII-mediated RyR phosphorylation. Altered Ca2+ homeostasis also increased signaling via both calcineurin/NFAT and extracellular signal-regulated kinase pathways. Altered myofilament Ca2+ buffering is the primary initiator of signaling cascades, indicating that directly targeting myofilament Ca2+ sensitivity provides an attractive therapeutic approach in HCM.

Dataset Information

Myofilament Ca sensitization increases cytosolic Ca binding affinity, alters intracellular Ca homeostasis, and causes pause-dependent Ca-triggered arrhythmia.

Rationale

Objective

Methods and results

Conclusions

Publications

Myofilament Ca sensitization increases cytosolic Ca binding affinity, alters intracellular Ca homeostasis, and causes pause-dependent Ca-triggered arrhythmia.

Similar Datasets

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