Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
German Center for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany.
Cells. 2024 Mar 8;13(6):476. doi: 10.3390/cells13060476.
The ubiquitous second messenger 3',5'-cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling (ECC) by signaling in discrete subcellular microdomains. Phosphodiesterase subfamilies 4B and 4D are critically involved in the regulation of cAMP signaling in mammalian cardiomyocytes. Alterations of PDE4 activity in human hearts has been shown to result in arrhythmias and heart failure. Here, we sought to systematically investigate specific roles of PDE4B and PDE4D in the regulation of cAMP dynamics in three distinct subcellular microdomains, one of them located at the caveolin-rich plasma membrane which harbors the L-type calcium channels (LTCCs), as well as at two sarco/endoplasmic reticulum (SR) microdomains centered around SR Ca-ATPase (SERCA2a) and cardiac ryanodine receptor type 2 (RyR2). Transgenic mice expressing Förster Resonance Energy Transfer (FRET)-based cAMP-specific biosensors targeted to caveolin-rich plasma membrane, SERCA2a and RyR2 microdomains were crossed to PDE4B-KO and PDE4D-KO mice. Direct analysis of the specific effects of both PDE4 subfamilies on local cAMP dynamics was performed using FRET imaging. Our data demonstrate that all three microdomains are differentially regulated by these PDE4 subfamilies. Whereas both are involved in cAMP regulation at the caveolin-rich plasma membrane, there are clearly two distinct cAMP microdomains at the SR formed around RyR2 and SERCA2a, which are preferentially controlled by PDE4B and PDE4D, respectively. This correlates with local cAMP-dependent protein kinase (PKA) substrate phosphorylation and arrhythmia susceptibility. Immunoprecipitation assays confirmed that PDE4B is associated with RyR2 along with PDE4D. Stimulated Emission Depletion (STED) microscopy of immunostained cardiomyocytes suggested possible co-localization of PDE4B with both sarcolemmal and RyR2 microdomains. In conclusion, our functional approach could show that both PDE4B and PDE4D can differentially regulate cardiac cAMP microdomains associated with calcium homeostasis. PDE4B controls cAMP dynamics in both caveolin-rich plasma membrane and RyR2 vicinity. Interestingly, PDE4B is the major regulator of the RyR2 microdomain, as opposed to SERCA2a vicinity, which is predominantly under PDE4D control, suggesting a more complex regulatory pattern than previously thought, with multiple PDEs acting at the same location.
普遍存在的第二信使 3',5'-环腺苷酸 (cAMP) 通过在离散的亚细胞微域中信号转导来调节心脏兴奋-收缩偶联 (ECC)。磷酸二酯酶亚家族 4B 和 4D 对于调节哺乳动物心肌细胞中的 cAMP 信号至关重要。已经证明，人心脏中 PDE4 活性的改变会导致心律失常和心力衰竭。在这里，我们试图系统地研究 PDE4B 和 PDE4D 在调节三个不同亚细胞微域中 cAMP 动力学中的特定作用，其中一个位于富含窖蛋白的质膜上，该质膜含有 L 型钙通道 (LTCCs)，以及两个围绕肌浆网 Ca-ATP 酶 (SERCA2a) 和心脏兰尼碱受体 2 型 (RyR2) 的肌浆网/内质网 (SR) 微域。表达针对富含窖蛋白的质膜、SERCA2a 和 RyR2 微域的基于荧光共振能量转移 (FRET) 的 cAMP 特异性生物传感器的转基因小鼠与 PDE4B-KO 和 PDE4D-KO 小鼠杂交。使用 FRET 成像直接分析这两个 PDE4 亚家族对局部 cAMP 动力学的特定影响。我们的数据表明，这三个微域都受到这些 PDE4 亚家族的不同调节。虽然两者都参与富含窖蛋白的质膜的 cAMP 调节，但在围绕 RyR2 和 SERCA2a 形成的 SR 中显然存在两个不同的 cAMP 微域，它们分别由 PDE4B 和 PDE4D 优先控制。这与局部 cAMP 依赖性蛋白激酶 (PKA) 底物磷酸化和心律失常易感性相关。免疫沉淀测定证实 PDE4B 与 PDE4D 一起与 RyR2 相关。免疫染色的心肌细胞的受激发射损耗 (STED) 显微镜检查表明 PDE4B 可能与肌浆膜和 RyR2 微域共定位。总之，我们的功能方法可以表明，PDE4B 和 PDE4D 都可以差异调节与钙稳态相关的心脏 cAMP 微域。PDE4B 控制富含窖蛋白的质膜和 RyR2 附近的 cAMP 动力学。有趣的是，PDE4B 是 RyR2 微域的主要调节剂，而不是 SERCA2a 附近，SERCA2a 附近主要受 PDE4D 控制，这表明比以前认为的更复杂的调节模式，多个 PDE 作用于同一位置。
