During physical exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in protein kinase A (PKA)–mediated phosphorylation of the cardiac ryanodine receptor RyR2. PKA-dependent “hyperphosphorylation” of the RyR2 channel has been proposed as a major impairment that contributes to progression of heart failure. However, the sites of PKA phosphorylation and their phosphorylation status in cardiac diseases are not well defined. Among the known RyR2 phosphorylation sites, serine 2030 (S2030) remains highly controversial as a site of functional impact. We examined the contribution of RyR2-S2030 to Ca²⁺ signaling and excitation–contraction coupling (ECC) in a transgenic mouse with an ablated RyR2-S2030 phosphorylation site (RyR2-S2030A^+/+). We assessed ECC gain by using whole-cell patch–clamp recordings and confocal Ca²⁺ imaging during β-ARs stimulation with isoproterenol (Iso) and consistent SR Ca²⁺ loading and L-type Ca²⁺ current (I_Ca) triggering. Under these conditions, ECC gain is diminished in mutant compared with WT cardiomyocytes. Resting Ca²⁺ spark frequency (CaSpF) with Iso is also reduced by mutation of S2030. In permeabilized cells, when SR Ca²⁺ pump activity is kept constant (using 2D12 antibody against phospholamban), cAMP does not change CaSpF in S2030A^+/+ myocytes. Using Ca²⁺ spark recovery analysis, we found that mutant RyR Ca²⁺ sensitivity is not enhanced by Iso application, contrary to WT RyRs. Furthermore, ablation of RyR2-S2030 prevents acceleration of Ca²⁺ waves and increases latency to the first spontaneous Ca²⁺ release after a train of stimulations during Iso treatment. Together, these results suggest that phosphorylation at S2030 may represent an important step in the modulation of RyR2 activity during β-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs. Graphical Abstract

Store-operated Ca²⁺entry (SOCE) is activated following depletion of the inositol-1,4,5-trisphosphate (InsP₃)-sensitive Ca²⁺pool to regulate proliferation in immortalized cell lines established from either primary or metastatic lesions. The molecular nature of SOCE may involve both Stim1, which senses Ca²⁺levels within the endoplasmic reticulum (ER) Ca²⁺reservoir, and a number of a Ca²⁺-permeable channels on the plasma membrane, including Orai1, Orai3, and members of the canonical transient receptor (TRPC1–7) family of ion channels. The present study was undertaken to assess whether SOCE is expressed and controls proliferation in primary cultures isolated from secondary lesions of heavily pretreated metastatic renal cell carcinoma (mRCC) patients. SOCE was induced following pharmacological depletion of the ER Ca²⁺store, but not by InsP₃-dependent Ca²⁺release. Metastatic RCC cells express Stim1-2, Orai1–3, and TRPC1–7 transcripts and proteins. In these cells, SOCE was insensitive to BTP-2, 10 µM Gd³⁺and Pyr6, while it was inhibited by 100 µM Gd³⁺, 2-APB, and carboxyamidotriazole (CAI). Neither Gd³⁺nor 2-APB or CAI impaired mRCC cell proliferation. Consistently, no detectable Ca²⁺signal was elicited by growth factor stimulation. Therefore, a functional SOCE is expressed but does not control proliferation of mRCC cells isolated from patients resistant to multikinase inhibitors.

Hypoxia plays a crucial role in acute and chronic renal injury, which is attributable to renal tubular and glomerular cell damage. Some studies provide evidence that hypoxia-dependent upregulation of the mitochondrial enzyme arginase type-II (Arg-II) in tubular cells promotes renal tubular injury. It is, however, not known whether Arg-II is also expressed in glomerular cells, particularly podocytes under hypoxic conditions, contributing to hypoxia-induced podocyte injury. The effects of hypoxia on human podocyte cells (AB8/13) in cultures and on isolated kidneys from wild-type (wt) and arg-ii gene-deficient (arg-ii^−/−) mice ex vivo, as well as on mice of the two genotypes in vivo, were investigated, respectively. We found that the Arg-II levels were enhanced in cultured podocytes in a time-dependent manner over 48 h, which was dependent on the stabilization of hypoxia-inducible factor 1α (HIF1α). Moreover, a hypoxia-induced derangement of cellular actin cytoskeletal fibers, a decrease in podocin, and an increase in mitochondrial ROS (mtROS) generation—as measured by MitoSOX—were inhibited by adenoviral-mediated arg-ii gene silencing. These effects of hypoxia on podocyte injury were mimicked by the HIFα stabilizing drug DMOG, which inhibits prolyl hydroxylases (PHD), the enzymes involved in HIFα degradation. The silencing of arg-ii prevented the detrimental effects of DMOG on podocytes. Furthermore, the inhibition of mtROS generation by rotenone—the inhibitor of respiration chain complex-I—recapitulated the protective effects of arg-ii silencing on podocytes under hypoxic conditions. Moreover, the ex vivo experiments with isolated kidney tissues and the in vivo experiments with mice exposed to hypoxic conditions showed increased Arg-II levels in podocytes and decreased podocyte markers regarding synaptopodin in wt mice but not in arg-ii^−/− mice. While age-associated albuminuria was reduced in the arg-ii^−/− mice, the hypoxia-induced increase in albuminuria was, however, not significantly affected in the arg-ii^−/−. Our study demonstrates that Arg-II in podocytes promotes cell injury. Arg-ii ablation seems insufficient to protect mice in vivo against a hypoxia-induced increase in albuminuria, but it does reduce albuminuria in aging.