L-type Ca 2؉ currents determine the shape of cardiac action potentials (AP) and the magnitude of the myoplasmic Ca 2؉ signal, which regulates the contraction force. The auxiliary Ca 2؉ channel subunits ␣2␦-1 and ␤2 are important regulators of membrane expression and current properties of the cardiac Ca 2؉ channel (CaV1.2). However, their role in cardiac excitation-contraction coupling is still elusive. Here we addressed this question by combining siRNA knockdown of the ␣2␦-1 subunit in a muscle expression system with simulation of APs and Ca 2؉ transients by using a quantitative computer model of ventricular myocytes. Reconstitution of dysgenic muscle cells with CaV1.2 (GFP-␣1C) recapitulates key properties of cardiac excitation-contraction coupling. Concomitant depletion of the ␣2␦-1 subunit did not perturb membrane expression or targeting of the pore-forming GFP-␣1C subunit into junctions between the outer membrane and the sarcoplasmic reticulum. However, ␣2␦-1 depletion shifted the voltage dependence of Ca 2؉ current activation by 9 mV to more positive potentials, and it slowed down activation and inactivation kinetics approximately 2-fold. Computer modeling revealed that the altered voltage dependence and current kinetics exert opposing effects on the function of ventricular myocytes that in total cause a 60% prolongation of the AP and a 2-fold increase of the myoplasmic Ca 2؉ concentration during each contraction. Thus, the Ca 2؉ channel ␣2␦-1 subunit is not essential for normal Ca 2؉ channel targeting in muscle but is a key determinant of normal excitation and contraction of cardiac muscle cells, and a reduction of ␣2␦-1 function is predicted to severely perturb normal heart function.calcium ͉ heart ͉ action potential ͉ dysgenic myotube L -type Ca 2ϩ currents occupy a key position for normal function and dysfunction of the heart. They contribute to the shape and duration of the cardiac action potential (AP) and to the cytoplasmic Ca 2ϩ transients, which activate contraction. Mutations in the cardiac voltage-gated Ca 2ϩ channel (Ca V 1.2) cause long QT syndrome and life-threatening arrhythmias (1, 2), and the Ca 2ϩ channels are critical mediators of both the therapeutic actions and undesirable side effects of frequently used drugs (3).Voltage-gated Ca 2ϩ channels are composed of a pore-forming ␣ 1 subunit and auxiliary ␣ 2 ␦, ␤, and ␥ subunits. The auxiliary subunits determine the expression levels and biophysical properties of the channels and thus participate in the central role of the Ca 2ϩ channel in cardiac excitation-contraction (EC) coupling. The importance of these subunits is underscored by the fact that attempts to generate knockout mice of the specific cardiac isoforms resulted in embryonic lethal phenotypes because of cardiac dysfunction (4, 5) (J. Offord, personal communication). Besides showing the essential role of the auxiliary channel subunits, the lack of viable knockout models hampered the analysis of their specific roles in shaping the cardiac AP and Ca 2ϩ transients.The ␣ 2 ␦ subunit is...

Podocytes play a critical role in glomerular barrier function, both in health and disease. However, in vivo terminally differentiated podocytes are difficult to be maintained in in vitro culture. Induced pluripotent stem cells (iPSCs) offer the unique possibility for directed differentiation into mature podocytes. The current differentiation protocol to generate iPSC-derived podocyte-like cells provides a robust and reproducible method to obtain podocyte-like cells after 10 days that can be employed in in vitro research and biomedical engineering. Previous published protocols were improved by testing varying differentiation media, growth factors, seeding densities, and time course conditions. Modifications were made to optimize and simplify the one-step differentiation procedure. In contrast to earlier protocols, adherent cells for differentiation were used, the use of fetal bovine serum (FBS) was reduced to a minimum, and thus ß-mercaptoethanol could be omitted. The plating densities of iPSC stocks as well as the seeding densities for differentiation cultures turned out to be a crucial parameter for differentiation results. Conditionally immortalized human podocytes served as reference controls. iPSC-derived podocyte-like cells showed a typical podocyte-specific morphology and distinct expression of podocyte markers synaptopodin, podocin, nephrin and WT-1 after 10 days of differentiation as assessed by immunofluorescence staining or Western blot analysis. qPCR results showed a downregulation of pluripotency markers Oct4 and Sox-2 and a 9-fold upregulation of the podocyte marker synaptopodin during the time course of differentiation. Cultured podocytes exhibited endocytotic uptake of albumin. In toxicological assays, matured podocytes clearly responded to doxorubicin (Adriamycin™) with morphological alterations and a reduction in cell viability after 48 h of incubation.

The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Aging, certain diseases and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). Here, we report the differentiation and characterization of iPSC lines into proximal tubular-like cells (PTL). The protocol is a step wise exposure of small molecules and growth factors, including the GSK3 inhibitor (CHIR99021), the retinoic acid receptor activator (TTNPB), FGF9 and EGF, to drive iPSC to PTL via cell stages representing characteristics of early stages of renal development. Genome-wide RNA sequencing showed that PTL clustered within a kidney phenotype. PTL expressed proximal tubular-specific markers, including megalin (LRP2), showed a polarized phenotype, and were responsive to parathyroid hormone. PTL could take up albumin and exhibited ABCB1 transport activity. The phenotype was stable for up to 7 days and was maintained after passaging. This protocol will form the basis of an optimized strategy for molecular investigations using iPSC derived PTL.

The skeletal muscle dihydropyridine receptor is a slowly-activating calcium channel that functions as the voltage sensor in excitation-contraction coupling. In addition to the pore-forming alpha(1S) subunit it contains the transmembrane alpha(2)delta-1 and gamma(1) subunits and the cytoplasmic beta(1a) subunit. Although the roles of the auxiliary subunits in calcium channel function have been intensively studied in heterologous expression systems, their functions in excitation-contraction coupling has only recently been elucidated in muscle cells of various null-mutant animal models. In this article we will briefly outline the current state of these investigations.