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MCU overexpression evokes disparate dose-dependent effects on mito-ROS and spontaneous Ca²⁺ release in hypertrophic rat cardiomyocytes.

ABSTRACT: Cardiac dysfunction in heart failure (HF) and diabetic cardiomyopathy (DCM) is associated with aberrant intracellular Ca²⁺ handling and impaired mitochondrial function accompanied with reduced mitochondrial calcium concentration (mito-[Ca²⁺]). Pharmacological or genetic facilitation of mito-Ca²⁺ uptake was shown to restore Ca²⁺ transient amplitude in DCM and HF, improving contractility. However, recent reports suggest that pharmacological enhancement of mito-Ca²⁺ uptake can exacerbate ryanodine receptor-mediated spontaneous sarcoplasmic reticulum (SR) Ca²⁺ release in ventricular myocytes (VMs) from diseased animals, increasing propensity to stress-induced ventricular tachyarrhythmia. To test whether chronic recovery of mito-[Ca²⁺] restores systolic Ca²⁺ release without adverse effects in diastole, we overexpressed mitochondrial Ca²⁺ uniporter (MCU) in VMs from male rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Measurement of mito-[Ca²⁺] using genetic probe mtRCamp1h revealed that mito-[Ca²⁺] in TAB VMs paced at 2 Hz under β-adrenergic stimulation is lower compared with shams. Adenoviral 2.5-fold MCU overexpression in TAB VMs fully restored mito-[Ca²⁺]. However, it failed to improve cytosolic Ca²⁺ handling and reduce proarrhythmic spontaneous Ca²⁺ waves. Furthermore, mitochondrial-targeted genetic probes MLS-HyPer7 and OMM-HyPer revealed a significant increase in emission of reactive oxygen species (ROS) in TAB VMs with 2.5-fold MCU overexpression. Conversely, 1.5-fold MCU overexpression in TABs, that led to partial restoration of mito-[Ca²⁺], reduced mitochondria-derived reactive oxygen species (mito-ROS) and spontaneous Ca²⁺ waves. Our findings emphasize the key role of elevated mito-ROS in disease-related proarrhythmic Ca²⁺ mishandling. These data establish nonlinear mito-[Ca²⁺]/mito-ROS relationship, whereby partial restoration of mito-[Ca²⁺] in diseased VMs is protective, whereas further enhancement of MCU-mediated Ca²⁺ uptake exacerbates damaging mito-ROS emission.NEW & NOTEWORTHY Defective intracellular Ca²⁺ homeostasis and aberrant mitochondrial function are common features in cardiac disease. Here, we directly compared potential benefits of mito-ROS scavenging and restoration of mito-Ca²⁺ uptake by overexpressing MCU in ventricular myocytes from hypertrophic rat hearts. Experiments using novel mito-ROS and Ca²⁺ biosensors demonstrated that mito-ROS scavenging rescued both cytosolic and mito-Ca²⁺ homeostasis, whereas moderate and high MCU overexpression demonstrated disparate effects on mito-ROS emission, with only a moderate increase in MCU being beneficial.

SUBMITTER: Hamilton S

PROVIDER: S-EPMC8794228 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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MCU overexpression evokes disparate dose-dependent effects on mito-ROS and spontaneous Ca2+ release in hypertrophic rat cardiomyocytes.

Hamilton Shanna S Terentyeva Radmila R Perger Fruzsina F Hernández Orengo Benjamín B Martin Benjamin B Gorr Matthew W MW Belevych Andriy E AE Clements Richard T RT Györke Sandor S Terentyev Dmitry D

American journal of physiology. Heart and circulatory physiology 20210820 4

Cardiac dysfunction in heart failure (HF) and diabetic cardiomyopathy (DCM) is associated with aberrant intracellular Ca2+ handling and impaired mitochondrial function accompanied with reduced mitochondrial calcium concentration (mito-[Ca2+]). Pharmacological or genetic facilitation of mito-Ca2+ uptake was shown to restore Ca2+ transient amplitude in DCM and HF, improving contractility. However, recent reports suggest that pharmacological enhancement o ...[more]

PMID: 34415186

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Project description:Spontaneous Ca2+-release events (SCaEs) from the sarcoplasmic reticulum play crucial roles in the initiation of cardiac arrhythmias by promoting triggered activity. However, the subcellular determinants of these SCaEs remain incompletely understood. Structural differences between atrial and ventricular cardiomyocytes, e.g., regarding the density of T-tubular membrane invaginations, may influence cardiomyocyte Ca2+-handling and the distribution of cardiac ryanodine receptors (RyR2) has recently been shown to undergo remodeling in atrial fibrillation. These data suggest that the subcellular distribution of Ca2+-handling proteins influences proarrhythmic Ca2+-handling abnormalities. Here, we employ computational modeling to provide an in-depth analysis of the impact of variations in subcellular RyR2 and L-type Ca2+-channel distributions on Ca2+-transient properties and SCaEs in a human atrial cardiomyocyte model. We incorporate experimentally observed RyR2 expression patterns and various configurations of axial tubules in a previously published model of the human atrial cardiomyocyte. We identify an increased SCaE incidence for larger heterogeneity in RyR2 expression, in which SCaEs preferentially arise from regions of high local RyR2 expression. Furthermore, we show that the propagation of Ca2+ waves is modulated by the distance between RyR2 bands, as well as the presence of experimentally observed RyR2 clusters between bands near the lateral membranes. We also show that incorporation of axial tubules in various amounts and locations reduces Ca2+-transient time to peak. Furthermore, selective hyperphosphorylation of RyR2 around axial tubules increases the number of spontaneous waves. Finally, we present a novel model of the human atrial cardiomyocyte with physiological RyR2 and L-type Ca2+-channel distributions that reproduces experimentally observed Ca2+-handling properties. Taken together, these results significantly enhance our understanding of the structure-function relationship in cardiomyocytes, identifying that RyR2 and L-type Ca2+-channel distributions have a major impact on systolic Ca2+ transients and SCaEs.

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.

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MCU overexpression evokes disparate dose-dependent effects on mito-ROS and spontaneous Ca²⁺ release in hypertrophic rat cardiomyocytes.

Publications

MCU overexpression evokes disparate dose-dependent effects on mito-ROS and spontaneous Ca<sup>2+</sup> release in hypertrophic rat cardiomyocytes.

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