Andrew M. Bellinger

Remodeling of ryanodine receptor complex causes “leaky” channels: A molecular mechanism for decreased exercise capacity

During exercise, defects in calcium (Ca2+) release have been proposed to impair muscle function. Here, we show that during exercise in mice and humans, the major Ca2+ release channel required for excitation–contraction coupling (ECC) in skeletal muscle, the ryanodine receptor (RyR1), is progressively PKA-hyperphosphorylated, S-nitrosylated, and depleted of the phosphodiesterase PDE4D3 and the RyR1 stabilizing subunit calstabin1 (FKBP12), resulting in “leaky” channels that cause decreased exercise tolerance in mice. Mice with skeletal muscle-specific calstabin1 deletion or PDE4D deficiency exhibited significantly impaired exercise capacity. A small molecule (S107) that prevents depletion of calstabin1 from the RyR1 complex improved force generation and exercise capacity, reduced Ca2+-dependent neutral protease calpain activity and plasma creatine kinase levels. Taken together, these data suggest a possible mechanism by which Ca2+ leak via calstabin1-depleted RyR1 channels leads to defective Ca2+ signaling, muscle damage, and impaired exercise capacity.

Stressed out: the skeletal muscle ryanodine receptor as a target of stress

Over the past century, understanding the mechanisms underlying muscle fatigue and weakness has been the focus of much investigation. However, the dominant theory in the field, that lactic acidosis causes muscle fatigue, is unlikely to tell the whole story. Recently, dysregulation of sarcoplasmic reticulum (SR) Ca(2+) release has been associated with impaired muscle function induced by a wide range of stressors, from dystrophy to heart failure to muscle fatigue. Here, we address current understandings of the altered regulation of SR Ca(2+) release during chronic stress, focusing on the role of the SR Ca(2+) release channel known as the type 1 ryanodine receptor.

Protein Kinase G Phosphorylates Cav1.2 α1c and β2 Subunits

Voltage-dependent Ca2+ channel function (Cav1.2, L-type Ca2+ channel) is required for cardiac excitation-contraction (E-C) coupling. Cav1.2 plays a key role in modulating cardiac function in response to classic signaling pathways, such as the renin-angiotensin system and sympathetic nervous system. Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with Cav1.2 current through the actions of cAMP and cGMP. Cardiac cGMP, which activates protein kinase G (PKG), is regulated by nitric oxide (NO), and natriuretic peptides. Although PKG has been reported to activate or inhibit Cav1.2 function, it is still unclear whether Cav1.2 subunits are PKG substrates. We have identified phosphorylation sites within the α1c and β2a subunits that are phosphorylated by PKGIα in vitro. We demonstrate that a subset of these phosphorylation sites is modulated, in a cGMP-PKG–specific manner, in intact HEK cells heterologously expressing α1c and β2a subunits. Using phospho-epitope–specific antibodies, we show that the phosphorylation of these residues is enhanced by PKG in intact cardiac myocytes. Activation of PKG in HEK cells transfected with α1c and β2a subunits caused an inhibition of Cav1.2 whole-cell current. PKG-mediated inhibition of Cav1.2 current was significantly reduced by coexpression of an alanine-substituted Cav1.2 β2a subunit (Ser496). Our results identify a molecular mechanism by which cGMP-PKG regulates Cav1.2 phosphorylation and function.

Protein kinase G phosphorylates Cav1.2 alpha1c and beta2 subunits.

Voltage-dependent Ca2+ channel function (Cav1.2, L-type Ca2+ channel) is required for cardiac excitation-contraction (E-C) coupling. Cav1.2 plays a key role in modulating cardiac function in response to classic signaling pathways, such as the renin-angiotensin system and sympathetic nervous system. Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with Cav1.2 current through the actions of cAMP and cGMP. Cardiac cGMP, which activates protein kinase G (PKG), is regulated by nitric oxide (NO), and natriuretic peptides. Although PKG has been reported to activate or inhibit Cav1.2 function, it is still unclear whether Cav1.2 subunits are PKG substrates. We have identified phosphorylation sites within the α1c and β2a subunits that are phosphorylated by PKGIα in vitro. We demonstrate that a subset of these phosphorylation sites is modulated, in a cGMP-PKG–specific manner, in intact HEK cells heterologously expressing α1c and β2a subunits. Using phospho-epitope–specific antibodies, we show that the phosphorylation of these residues is enhanced by PKG in intact cardiac myocytes. Activation of PKG in HEK cells transfected with α1c and β2a subunits caused an inhibition of Cav1.2 whole-cell current. PKG-mediated inhibition of Cav1.2 current was significantly reduced by coexpression of an alanine-substituted Cav1.2 β2a subunit (Ser496). Our results identify a molecular mechanism by which cGMP-PKG regulates Cav1.2 phosphorylation and function.

Nonshivering thermogenesis protects against defective calcium handling in muscle

When acutely exposed to a cold environment, mammals shiver to generate heat. During prolonged cold exposure, shivering is replaced by adaptive adrenergic nonshivering thermogenesis with increased heat production in brown adipose tissue due to activation of uncoupling protein‐1 (UGP1). This cold acclimation is associated with chronically increased sympathetic stimulation of skeletal muscle, which may increase the sarcoplasmic reticulum (SR) Ca2+ leak via destabilized ryanodine receptor 1 (RyR1) channel complexes. Here, we use genetically engineered UGP1‐deficient (UGP1‐KO) mice that rely completely on shivering in the cold. We examine soleus muscle, which participates in shivering, and flexor digitorum brevis (FDB) muscle, a distal and superficial muscle that does not shiver. Soleus muscles of cold‐acclimated UGP1‐KO mice exhibited severe RyR1 PKA hyperphosphorylation and calstabin1 depletion, as well as markedly decreased SR Ca2+ release and force during contractions. In stark contrast, the RyR1 channel complexes were little affected, and Ca2+ and force were not decreased in FDB muscles of cold‐acclimated UGP1‐KO mice. These results indicate that activation of UGP1‐mediated heat production in brown adipose tissue during cold exposure reduces the necessity for shivering and thus prevents the development of severe dysfunction in shivering muscles.— Aydin, J., Shabalina, I. G., Place, N., Reiken, S., Zhang, S.‐J., Bellinger, A. M., Nedergaard, J., Cannon, B., Marks, A. R., Bruton, J. D., Westerblad, H. Nonshivering thermogenesis protects against defective calcium handling in muscle. FASEB J. 22, 3919–3924 (2008)

Protein Kinase G Phosphorylates Ca v 1.2 α 1c and β 2 Subunits

Voltage-dependent Ca2+ channel function (Cav1.2, L-type Ca2+ channel) is required for cardiac excitation-contraction (E-C) coupling. Cav1.2 plays a key role in modulating cardiac function in response to classic signaling pathways, such as the renin-angiotensin system and sympathetic nervous system. Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with Cav1.2 current through the actions of cAMP and cGMP. Cardiac cGMP, which activates protein kinase G (PKG), is regulated by nitric oxide (NO), and natriuretic peptides. Although PKG has been reported to activate or inhibit Cav1.2 function, it is still unclear whether Cav1.2 subunits are PKG substrates. We have identified phosphorylation sites within the α1c and β2a subunits that are phosphorylated by PKGIα in vitro. We demonstrate that a subset of these phosphorylation sites is modulated, in a cGMP-PKG–specific manner, in intact HEK cells heterologously expressing α1c and β2a subunits. Using phospho-epitope–specific antibodies, we show that the phosphorylation of these residues is enhanced by PKG in intact cardiac myocytes. Activation of PKG in HEK cells transfected with α1c and β2a subunits caused an inhibition of Cav1.2 whole-cell current. PKG-mediated inhibition of Cav1.2 current was significantly reduced by coexpression of an alanine-substituted Cav1.2 β2a subunit (Ser496). Our results identify a molecular mechanism by which cGMP-PKG regulates Cav1.2 phosphorylation and function.