Dataset Information

The β2-subunit of voltage-gated calcium channels inhibits cardiomyocyte hypertrophy through a channel-independent mechanism.

ABSTRACT: L-type voltage-gated calcium channels (LTCCs) regulate crucial physiological processes in the heart. They are composed of the Cav1 pore-forming subunit and the accessory subunits Cav, Cav2 and Cav. Cav is a cytosolic soluble protein that regulates channel trafficking and activity, but it also exerts other LTCC-independent functions. Cardiac hypertrophy, a relevant risk factor for the development of congestive heart failure, depends on the activation of calcium-dependent pro-hypertrophic signaling cascades; however, the role of LTCCs in this pathology remains controversial. Here, by using shRNA-mediated Cav silencing, we demonstrate that Cav2 downregulation enhances 1-adrenergic receptor agonist-induced cardiomyocyte hypertrophy in an LTCC-independent manner. We report that a pool of Cav2 is targeted to the nucleus in cardiomyocytes and that the expression of this nuclear fraction decreases during in vitro and in vivo induction of cardiac hypertrophy. Moreover, the overexpression of nucleus-targeted Cav2 in cardiomyocytes inhibits in vitro-induced hypertrophy. Quantitative proteomic analyses showed that Cav2 knockdown leads to changes in the expression of diverse myocyte proteins, including reduction of calpastatin, an endogenous inhibitor of the calcium-dependent protease calpain. Accordingly, Cav2-deficient cardiomyocytes had a two-fold increase in calpain activity as compared to control cells. Furthermore, inhibition of calpain activity in Cav2-deficient cells abolished the enhanced 1-adrenergic receptor agonist-induced hypertrophy observed in these cells. Our findings indicate that in cardiomyocytes, a nuclear pool of Cav2 participates in cellular functions that are independent of LTCC activity. They also indicate that a downregulation of nuclear Cav2 during cardiac hypertrophy promotes the activation of calpain-dependent hypertrophic pathways.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Rattus Norvegicus (rat) Rattus

TISSUE(S): Cell Culture

SUBMITTER: Katalin Barkovits

LAB HEAD: Katalin Barkovits

PROVIDER: PXD016483 | Pride | 2021-09-09

REPOSITORIES: Pride

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Publications

The β2-Subunit of Voltage-Gated Calcium Channels Regulates Cardiomyocyte Hypertrophy.

Pickel Simone S Cruz-Garcia Yiliam Y Bandleon Sandra S Barkovits Katalin K Heindl Cornelia C Völker Katharina K Abeßer Marco M Pfeiffer Kathy K Schaaf Alice A Marcus Katrin K Eder-Negrin Petra P Kuhn Michaela M Miranda-Laferte Erick E

Frontiers in cardiovascular medicine 20210707

L-type voltage-gated calcium channels (LTCCs) regulate crucial physiological processes in the heart. They are composed of the Cavα1 pore-forming subunit and the accessory subunits Cavβ, Cavα2δ, and Cavγ. Cavβ is a cytosolic protein that regulates channel trafficking and activity, but it also exerts other LTCC-independent functions. Cardiac hypertrophy, a relevant risk factor for the development of congestive heart failu ...[more]

PMID: 34307509

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Project description:Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persists for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods. Mouse embryonic fibroblasts were treated with different combinations of transcription factors to drive transdifferentiation to induced cardiomyocytes (iCMs). Putative iCMs were enriched by zeocin selection. Zeocin resistance was conferred to iCMs via the TroponinT-GCaMP5-Zeo lentiviral reporter.

Project description:Cardiac hypertrophy is an important and independent risk factor for the development of cardiac myopathy that may lead to heart failure. Cardiac hypertrophy manifests as an enlargement of the individual cardiomyocytes, which impairs the function of the heart. The only way to cure end-stage cardiac myopathy is by heart transplantation, a possibility limited due to lack of donor hearts. Therefore, early diagnosis of cardiac hypertrophy is needed in order to be able to initiate interventions that may prevent further progression of the disease. The mechanisms underlying the development of cardiac hypertrophy are yet not well understood. To increase the knowledge about mechanisms and regulatory pathways involved in the progression of cardiac hypertrophy, we have developed a human induced pluripotent stem cell (hiPSC)-based in vitro model of cardiac hypertrophy and performed extensive characterization of the model using multi-omics analyses. In a series of experiments, hiPSC-derived cardiomyocytes were stimulated with Endothelin-1 for 8, 24, 48 and 72 hours and their transcriptome and secreted proteome were analyzed thoroughly. The transcriptomic data show many enriched canonical pathways related to cardiac hypertrophy already at the earliest time point, e.g., cardiac hypertrophy signaling, actin cytoskeleton signaling and PI3K/AKT signaling. Cluster analysis of the differentially expressed genes showed that there are numerous clusters of genes that are dysregulated over the time period of 8 to 72h. An integrated transcriptome-secretome analysis enabled the identification of multimodal biomarkers of high relevance for monitoring early cardiac hypertrophy progression. Taken together, the results from this study demonstrate that our in vitro model displays a hypertrophic response on transcriptomic- and secreted proteomic level. The results also provide novel insight into the underlying mechanisms of cardiac hypertrophy and novel putative early cardiac hypertrophy biomarkers have been identified that will be further validated to assess their clinical relevance.

Dataset Information

The β2-subunit of voltage-gated calcium channels inhibits cardiomyocyte hypertrophy through a channel-independent mechanism.

Publications

The β<sub>2</sub>-Subunit of Voltage-Gated Calcium Channels Regulates Cardiomyocyte Hypertrophy.

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