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Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes
Published on: September 18, 2017
Calcium Handling Remodeling Underlies Impaired Sympathetic Stress Response in Ventricular Myocardium from Cacna1c
Hauke Fender1, Kim Walter1, Aytug K Kiper2,3
1Institute of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Biochemical and Pharmacological Center (BPC) Marburg, University of Marburg, 35032 Marburg, Germany.
Insights
Haploinsufficient Cacna1c rats show normal basal cardiac function but impaired calcium handling under stress. This is due to altered calcium handling proteins and reduced RyR2 phosphorylation, impacting cardiac response to beta-adrenergic stimulation.
Area of Science:
- Cardiovascular Physiology
- Molecular Cardiology
- Genetics of Cardiac Disease
Background:
- CACNA1C gene mutations are linked to neuropsychiatric and cardiac conditions.
- Haploinsufficient Cacna1c rats exhibit a behavioral phenotype; their cardiac phenotype remains uncharacterized.
- Understanding the cardiac implications of Cacna1c deficiency is crucial for disease modeling.
Purpose of the Study:
- To elucidate the cardiac phenotype of Cacna1c-deficient rats, focusing on cellular calcium handling.
- To investigate the impact of Cacna1c deficiency on cardiomyocyte function under basal and stimulated conditions.
- To identify alterations in calcium handling proteins and their phosphorylation states.
Main Methods:
- Isolated ventricular myocyte electrophysiology and calcium transient measurements.
- Immunoblotting to assess protein expression and phosphorylation levels (Cav1.2, SERCA2a, NCX, RyR2).
- Langendorff-perfused heart studies to evaluate cardiac function and signaling pathways.
- Pharmacological stimulation with isoprenaline to mimic sympathetic stress.
Main Results:
- Cacna1c myocytes showed normal basal L-type Ca2+ current, Ca2+ transients, SR Ca2+ load, and contractility.
- Basal LV tissue revealed reduced Cav1.2, increased SERCA2a and NCX, and augmented RyR2 phosphorylation (S2808).
- Isoprenaline stimulation impaired CaT amplitude and fractional shortening, with reduced potency and efficacy in Cacna1c myocytes.
- Sarcolemmal Ca2+ influx and fractional SR Ca2+ release were diminished post-isoprenaline in Cacna1c myocytes.
- Isoprenaline-induced RyR2 phosphorylation (S2808, S2814) was attenuated in Cacna1c hearts.
Conclusions:
- Cacna1c myocytes exhibit basal remodeling of calcium handling proteins despite normal function.
- Sympathetic stress unmasks impaired calcium influx, SR release, and CaTs in Cacna1c cardiomyocytes.
- Reduced RyR2 phosphorylation reserve contributes to the blunted response to beta-adrenergic stimulation in Cacna1c hearts.
Abstract:
CACNA1C encodes the pore-forming α1C subunit of the L-type Ca2+ channel, Cav1.2. Mutations and polymorphisms of the gene are associated with neuropsychiatric and cardiac disease. Haploinsufficient Cacna1c rats represent a recently developed model with a behavioral phenotype, but its cardiac phenotype is unknown. Here, we unraveled the cardiac phenotype of Cacna1c rats with a main focus on cellular Ca2+ handling mechanisms. Under basal conditions, isolated ventricular Cacna1c myocytes exhibited unaltered L-type Ca2+ current, Ca2+ transients (CaTs), sarcoplasmic reticulum (SR) Ca2+ load, fractional release, and sarcomere shortenings. However, immunoblotting of left ventricular (LV) tissue revealed reduced expression of Cav1.2, increased expression of SERCA2a and NCX, and augmented phosphorylation of RyR2 (at S2808) in Cacna1c rats. The β-adrenergic agonist isoprenaline increased amplitude and accelerated decay of CaTs and sarcomere shortenings in both Cacna1c and WT myocytes. However, the isoprenaline effect on CaT amplitude and fractional shortening (but not CaT decay) was impaired in Cacna1c myocytes exhibiting both reduced potency and efficacy. Moreover, sarcolemmal Ca2+ influx and fractional SR Ca2+ release after treatment with isoprenaline were smaller in Cacna1c than in WT myocytes. In Langendorff-perfused hearts, the isoprenaline-induced increase in RyR2 phosphorylation at S2808 and S2814 was attenuated in Cacna1c compared to WT hearts. Despite unaltered CaTs and sarcomere shortenings, Cacna1c myocytes display remodeling of Ca2+ handling proteins under basal conditions. Mimicking sympathetic stress with isoprenaline unmasks an impaired ability to stimulate Ca2+ influx, SR Ca2+ release, and CaTs caused, in part, by reduced phosphorylation reserve of RyR2 in Cacna1c cardiomyocytes.
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