Orai1 and Stim1 regulate normal and hypertrophic growth in cardiomyocytes

doi:10.1016/j.yjmcc.2010.01.020

Journal of Molecular and Cellular Cardiology

Volume 48, Issue 6, June 2010, Pages 1329-1334

https://doi.org/10.1016/j.yjmcc.2010.01.020 Get rights and content

Abstract

Cardiac hypertrophy is an independent risk for heart failure (HF) and sudden death. Deciphering signalling pathways dependent on extracellular calcium (Ca²⁺) influx that control normal and pathological cardiac growth may enable identification of novel therapeutic targets. The objective of the present study is to determine the role of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel Orai1 and stromal interaction molecule 1 (Stim1) in postnatal cardiomyocyte store operated Ca²⁺ entry (SOCE) and impact on normal and hypertrophic postnatal cardiomyocyte growth. Employing a combination of siRNA-mediated gene silencing, cultured neonatal rat ventricular cardiomyocytes together with indirect immunofluorescence, epifluorescent Ca²⁺ imaging and site-specific protein phosphorylation and real-time mRNA expression analysis, we show for the first time that both Orai1 and Stim1 are present in cardiomyocytes and required for SOCE due to intracellular Ca²⁺ store depletion by thapsigargin. Stim1-KD but not Orai1-KD significantly decreased diastolic Ca²⁺ levels and caffeine-releasable Ca²⁺ from the sarcoplasmic reticulum (SR). Conversely, Orai1-KD but not Stim1-KD significantly diminished basal NRCM cell size, anp and bnp mRNA levels and activity of the calcineurin (CnA) signalling pathway although diminishing both Orai1 and Stim1 proteins similarly attenuated calmodulin kinase II (CamKII) and ERK1/2 activity under basal conditions. Both Orai1- and Stim1-KD completely abrogated phenylephrine (PE) mediated hypertrophic NRCM growth and enhanced natriuretic factor expression by inhibiting G_q-protein conveyed activation of the CamKII and ERK1/2 signalling pathway. Interestingly, only Orai1-KD but not Stim1-KD prevented Gq-mediated CaN-dependent prohypertrophic signalling. This study shows for the first time that both Orai1 and Stim1 have a key role in cardiomyocyte SOCE regulating both normal and hypertrophic postnatal cardiac growth in vitro.

Introduction

Cardiac myocytes express a large number of Ca²⁺ signalling systems that are required to regulate many different cellular functions including physiological growth, contractile function and pathological hypertrophy [1]. Sustained Ca²⁺ signals are required to induce physiological postnatal and pathological cardiac hypertrophy via the CamK and the calcineurin-NFAT pathway, which are separate from the Ca²⁺ signals required for electric contraction coupling (ECC) [2].

Store operated Ca²⁺ entry (SOCE) is an important process in cellular physiology that controls such diverse functions as refilling of intracellular Ca²⁺ stores, activation of enzymatic activity and gene transcription. Recently the two key players of SOCE have been identified, Stim1 and Orai1. In several non-excitable cells but also in skeletal and smooth muscle cells it was shown that Stim1/Orai1 is responsible for SOCE [3]. Moreover, a recent study demonstrated for the first time the expression of Stim1 in cardiomyocytes and investigated a role of Stim1 in cardiac hypertrophy [4].

Stim1 is a 77 kDa single-pass transmembrane protein located primarily in the ER membrane. Stim1 proteins possess conserved N-terminal Ca²⁺ binding EF hands within the ER lumen, where they are thought to sense the luminal Ca²⁺ concentration. Orai1 is a small plasmamembrane protein of 32 kDa with four transmembrane domains and an amino carboxyl end that face the cytosol [5]. Orai1 was identified as the CRAC (Ca²⁺ release-activated Ca²⁺) channel pore-forming subunit protein. Ca²⁺ release by IP-3 Receptors causes Stim1 oligomerization and relocalisation into ER-plasma membrane junctions. This subsequently activates Orai1 in the adjacent plasma membrane and causes Ca²⁺ entry into the cytosol via CRAC that mediates Ca²⁺ signals required for activation of CamK and CnA.

However the exact function of Orai1 and Stim1 in cardiac muscle is unknown. In the present study we describe the existence of a Stim1/Orai1 signalling system in cardiac myocytes and provide evidence that both Orai1 and Stim1 have a key role in cardiomyocyte SOCE regulating both normal and hypertrophic postnatal cardiac growth in vitro.

Section snippets

Isolation and primary culture of neonatal rat ventricular cardiomyocytes

Ventricular cardiomyocytes from 1 to 2 day old rat neonatal hearts (NRCMs) were prepared by trypsin digestion as described previously [6]. After 24 h the medium was replaced by a 0.5% serum-containing DMEM and NRCM were transfected with different siRNAs. 48 h after transfection cells were stimulated with phenylephrine for up to 48 h. After indicated time points, cells were washed with PBS and either subjected to TRIZOL or lysis buffer (PBS pH 7.4, SDS 2%, 2 mM EGTA/EDTA) containing a mixture of 1%

Results and discussion

Since Orai1 and Stim1 orchestrate Ca²⁺-dependent regulation of skeletal muscle growth and key biological functions in non-cardiac cells due to SOCE, we first investigated expression levels both of Orai1 and Stim1 in isolated postnatal ventricular cardiomyocytes. Store operated Ca²⁺ Entry is one ubiquitous Ca²⁺ signalling pathway whereby depletion of intracellular Ca²⁺ stores activates Ca²⁺ channels in the plasma membrane. The Ca²⁺ entry via CRAC subsequently activates both CnA and CamKII

Acknowledgments

This study was supported by grants from the National Institute of Health (RO1 HL92130 and RO1HL92130-02S1 to P. Most), Deutsche Forschungsgemeinschaft (562/1-1 to P.Most and VO1659/1-1 to M.Voelkers) and Bundesministerium fuer Bildung und Forschung (01GU0572 to P.Most).

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The store-operated Ca²⁺ entry (SOCE) represents an important route for generating cellular Ca²⁺ signals that are implicated in physiological and various pathological scenarios that include diabetic cardiomyopathy (DM-CMP) which is well known to have Ca²⁺ dysregulation among other salient features. In this study, we investigated the role of SOCE in Ca²⁺ handling of cardiomyocytes obtained from adult male Wistar rats that were made diabetic by intraperitoneal administration of streptozotocin (STZ 50 mg/kg). We also included another group of rats with diabetes induced by STZ administration but received an angiotensin II receptor blocker - losartan. In whole cell recordings with isolated cardiomyocytes, the SOCE-representative whole-cell current I_CRAC was found to be significantly reduced for the diabetic group compared to the control group and chronic losartan treatment could restore I_CRAC to a level comparable to the control group. However, in contrast to the observed reduction in I_CRAC, Orai1 and Orai3 proteins were found to be significantly upregulated in diabetic condition whereas no significant change in the expression levels of Stim1, Stim2 and Orai2 was observed. Also, losartan treatment did not affect the expression pattern of these key proteins for SOCE in diabetic group. The observed imbalance between the functional read out of SOCE (peak I_CRAC size) and expression levels of the underlying proteins was puzzling but could be, among other possibilities, due to impairment of interaction between Stim and Orai proteins. We argue that the observed changes in SOCE with diabetes could be a contributing factor for the Ca²⁺ dyshomeostasis associated with diabetic cardiomyopathies and blockade of angiotensin II receptor may potentially restore normal SOCE in diabetic cardiomyocytes.
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Citation Excerpt :
Emerging evidence shows that STIM1 mediated store-operated Ca2+ entry (SOCE) plays a central role in cardiac hypertrophy and heart failure (Groschner et al., 2017). SOCE has also been demonstrated to be responsible for phenylephrine-induced cardiac hypertrophy (Voelkers et al., 2010) (Li et al., 2018) (Zhao et al., 2017). This is consistent with our data, especially the fact that YM58483 (an Orai1 channel blocker) abrogates PE-induced enhanced SOCE also alleviates PE-induced hypertrophy in NRCMs.
Trans-cinnamaldehyde (TCA) is a main compound of Cinnamomum cassia, used in traditional Chinese medicine to treat many ailments. Increasing evidence has demonstrated the therapeutic effects of TCA in cardiovascular diseases.
The present study aimed to determine whether TCA exerts antihypertrophic effects in vitro and in vivo and to elucidate the underlying mechanisms of these effects.
Neonatal rat cardiac myocytes (NRCMs) and adult mouse cardiac myocytes (AMCMs) were treated with 50 μΜ phenylephrine (PE) for 48 h. Tubulin detyrosination, store-operated Ca²⁺ entry (SOCE), stromal interaction molecule-1 (STIM1)/Orai1 translocation, and calcineurin/nuclear factor of activated T-cells (NFAT) signaling pathways were analyzed in NRCMs. Meanwhile, tubulin detyrosination, junctophilin-2, T-tubule distribution pattern, Ca²⁺ handling, and sarcomere shortening were observed in AMCMs. Male C57BL/6 mice were stimulated with PE (70 mg/kg per day) with or without TCA treatment for 2 weeks. Cardiac hypertrophy and tubulin detyrosination were also assessed.
TCA was confirmed to alleviate cardiac hypertrophy induced by PE stimulation in vitro and in vivo. PE-induced cardiac hypertrophy was associated with excessive tubulin detyrosination and overexpression of vasohibin 1 (VASH1) and small vasohibin binding protein (SVBP), two key proteins responsible for tubulin detyrosination. These effects were largely blocked by TCA administration. PE treatment also enhanced SOCE with massive translocation of STIM1 and Orai1, Ca²⁺ mishandling, reduced sarcomere shortening, junctophilin-2, and T-tubule redistribution, all of which were significantly ameliorated by TCA administration.
Our study indicated that the therapeutic effects of TCA against cardiac hypertrophy may be associated with its ability to reduce tubulin detyrosination.
Saraf-dependent activation of mTORC1 regulates cardiac growth
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Pathological cardiac hypertrophy is an independent risk for heart failure (HF) and sudden death. Deciphering signaling pathways regulating intracellular Ca²⁺ homeostasis that control adaptive and pathological cardiac growth may enable identification of novel therapeutic targets. The objective of the present study is to determine the role of the store-operated calcium entry-associated regulatory factor (Saraf), encoded by the Tmem66 gene, on cardiac growth control in vitro and in vivo. Saraf is a single-pass membrane protein located at the sarco/endoplasmic reticulum and regulates intracellular calcium homeostasis. We found that Saraf expression was upregulated in the hypertrophied myocardium and was sufficient for cell growth in response to neurohumoral stimulation. Increased Saraf expression caused cell growth, which was associated with dysregulation of calcium-dependent signaling and sarcoplasmic reticulum calcium content. In vivo, Saraf augmented cardiac myocyte growth in response to angiotensin II and resulted in increased cardiac remodeling together with worsened cardiac function. Mechanistically, Saraf activated mTORC1 (mechanistic target of rapamycin complex 1) and increased protein synthesis, while mTORC1 inhibition blunted Saraf-dependent cell growth. In contrast, the hearts of Saraf knockout mice and Saraf-deficient myocytes did not show any morphological or functional alterations after neurohumoral stimulation, but Saraf depletion resulted in worsened cardiac function after acute pressure overload. SARAF knockout blunted transverse aortic constriction cardiac myocyte hypertrophy and impaired cardiac function, demonstrating a role for SARAF in compensatory myocyte growth. Collectively, these results reveal a novel link between sarcoplasmic reticulum calcium homeostasis and mTORC1 activation that is regulated by Saraf.
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Store-operated Ca²⁺ entry (SOCE) is an essential Ca²⁺ signaling mechanism present in most animal cells. SOCE refers to Ca²⁺ influx that is activated by depletion of sarco/endoplasmic reticulum (S/ER) Ca²⁺ stores. The main components of SOCE are STIM and Orai. STIM proteins function as S/ER Ca²⁺ sensors, and upon S/ER Ca²⁺ depletion STIM rearranges to S/ER-plasma membrane junctions and activates Orai Ca²⁺ influx channels. Studies have implicated SOCE in cardiac hypertrophy pathogenesis, but SOCE's role in normal heart physiology remains poorly understood. We therefore analyzed heart-specific SOCE function in Drosophila, a powerful animal model of cardiac physiology. We show that heart-specific suppression of Stim and Orai in larvae and adults resulted in reduced contractility consistent with dilated cardiomyopathy. Myofibers were also highly disorganized in Stim and Orai RNAi hearts, reflecting possible decompensation or upregulated stress signaling. Furthermore, we show that reduced heart function due to SOCE suppression adversely affected animal viability, as heart specific Stim and Orai RNAi animals exhibited significant delays in post-embryonic development and adults died earlier than controls. Collectively, our results demonstrate that SOCE is essential for physiological heart function, and establish Drosophila as an important model for understanding the role of SOCE in cardiac pathophysiology.
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2024, Annals of Clinical and Laboratory Science

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Rapid communicationOrai1 and Stim1 regulate normal and hypertrophic growth in cardiomyocytes

Abstract

Introduction

Section snippets

Isolation and primary culture of neonatal rat ventricular cardiomyocytes

Results and discussion

Acknowledgments

Biochem Biophys Res Commun

J Biol Chem

J Biol Chem

Cardiac calcium signalling

Biochem Soc Trans

Cardiac hypertrophy: the good, the bad, and the ugly

Annu Rev Physiol

STIMulating store-operated Ca(2+) entry

Nat Cell Biol

Orai1 is an essential pore subunit of the CRAC channel

Nature

Cardiac adenoviral S100A1 gene delivery rescues failing myocardium

J Clin Invest

Rapid communication
Orai1 and Stim1 regulate normal and hypertrophic growth in cardiomyocytes