Caffeine-mediated alleviation of imbalance of calcium homeostasis and apoptosis in cardiomyocytes caused by RyR2 knockdown

doi:10.16150/j.1671-2870.2026.03.011

Abstract

Abstract:

Objective This study aims to investigate the effects of low-concentration caffeine on calcium homeostasis and apoptosis in rat cardiomyocytes with ryanodine receptor 2 (RyR2) knockdown. Methods Neonatal Sprague-Dawley (SD) rats of specific pathogen-free (SPF) grade were used, and neonatal rat ventricular myocytes (NRVMs) were isolated and extracted. Synthetic small interfering RNA (siRNA) targeting RyR2 and the negative control (NC) were transfected into NRVMs, and the cells were divided into four groups: NC group (transfected with siRNA NC), siRyR2 group (transfected with RyR2 siRNA), NC+caffeine group (0.2 mmol/L caffeine added after siRNA NC transfection), and siRyR2+caffeine group (0.2 mmol/L caffeine added after RyR2 siRNA transfection). The mRNA and protein expression levels of RyR2 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Cell apoptosis and calcium ions were detected by flow cytometry, and free calcium ion concentration was detected by confocal calcium ion imaging. Data from the NC, siRyR2, NC+caffeine, and siRyR2+caffeine groups were analyzed by one-way analysis of variance. Results Compared with the NC group, the mRNA and protein expression levels of RyR2 in the siRyR2 group were significantly decreased (mRNA: 0.41±0.04 vs 1.00±0.09, P=0.000 3; protein: 0.34±0.04 vs 0.72±0.02, P=0.000 1), calcium ion concentration was significantly decreased (flow cytometry: 0.80±0.02 vs 1.00±0.02, P<0.000 1; confocal: 61.08%±0.75% vs 100.00%±2.33%, P<0.000 1), and the apoptosis rate was significantly increased (9.21%±0.07% vs 4.19%±0.07%, P<0.000 1). In the NC+caffeine group, the mRNA and protein expression levels of RyR2 were significantly increased (mRNA: 1.27±0.15 vs 1.00±0.09, P=0.033 6; protein: 0.92±0.10 vs 0.72±0.02, P=0.006 5), calcium ion concentration was significantly increased (flow cytometry: 01.33±0.04 vs 1.00±0.02, P<0.000 1; confocal: 116.21%±1.51% vs 100.00%±2.33%, P=0.000 6), and the apoptosis rate was significantly decreased (2.97%±0.13% vs 4.19%±0.07%, P<0.000 1). Compared with the siRyR2 group, the mRNA and protein expression levels of RyR2 in the siRyR2+caffeine group were significantly increased (mRNA: 0.67±0.08 vs 0.41±0.04, P=0.044 7; protein: 0.54±0.01 vs 0.34±0.04, P=0.009 1), calcium ion concentration was significantly increased (flow cytometry: 0.87±0.02 vs 0.80±0.02, P=0.026 3; confocal: 79.14%±5.02% vs 61.08%±0.75%, P=0.000 3), and the apoptosis rate was significantly decreased (7.59%±0.10% vs 9.21%±0.07%, P<0.000 1). Compared with the NC+caffeine group, the mRNA and protein expression levels of RyR2 in the siRyR2+caffeine group were significantly decreased (mRNA: 0.67±0.08 vs 1.27±0.15, P=0.000 2; protein: 0.54±0.01 vs 0.92±0.10, P<0.000 1), calcium ion concentration was significantly decreased (flow cytometry: 0.87±0.02 vs 1.33±0.04, P<0.000 1; confocal: 79.14%±5.02% vs 116.21%±1.51%, P<0.000 1), and the apoptosis rate was significantly increased (7.59%±0.10% vs 2.97%±0.13%, P<0.000 1). Conclusions Low-concentration caffeine can promote calcium ion release, restore calcium homeostasis, and reduce apoptosis by upregulating RyR2 in cardiomyocytes. This interaction mechanism between caffeine and RyR2 can provide a reference for the development of therapeutic drugs for heart diseases such as arrhythmias caused by decreased RyR2 expression.

Key words: Cardiovascular diseases, Ryanodine receptor 2, Caffeine, Calcium homeostasis, Apoptosis

CLC Number:

R393
R541.7

YE Yuanzheng, FU Xiaoxiao, MEN Li, MA Qiancheng, ZHANG Andi. Caffeine-mediated alleviation of imbalance of calcium homeostasis and apoptosis in cardiomyocytes caused by RyR2 knockdown[J]. Journal of Diagnostics Concepts & Practice, 2026, 25(03): 346-353.

Figures/Tables 5

Table 1

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Name	Sequence	Product size（bp）
RyR2-F	5' -GATTCCAGCCAGATTGAG- 3'	110
RyR2-R	5' -TTGCCTATTGTTCCGTTT- 3'
GAPDH-F	5' -CAAGTTCAACGGCACAG- 3'	138
GAPDH-R	5' -CCAGTAGACTCCACGACAT -3'