Long QT syndrome (LQTS) is a genetic disease characterized by a prolonged QT interval in an electrocardiogram (ECG), leading to higher risk of sudden cardiac death. Among the 12 identified genes causal to heritable LQTS, ∼90% of affected individuals harbor mutations in either KCNQ1 or human ether-a-go-go related genes (hERG), which encode two repolarizing potassium currents known as I Ks and I Kr . The ability to quantitatively assess contributions of different current components is therefore important for investigating disease phenotypes and testing effectiveness of pharmacological modulation. Here we report a quantitative analysis by simulating cardiac action potentials of cultured human cardiomyocytes to match the experimental waveforms of both healthy control and LQT syndrome type 1 (LQT1) action potentials. The quantitative evaluation suggests that elevation of I Kr by reducing voltage sensitivity of inactivation, not via slowing of deactivation, could more effectively restore normal QT duration if I Ks is reduced. Using a unique specific chemical activator for I Kr that has a primary effect of causing a right shift of V 1/2 for inactivation, we then examined the duration changes of autonomous action potentials from differentiated human cardiomyocytes. Indeed, this activator causes dose-dependent shortening of the action potential durations and is able to normalize action potentials of cells of patients with LQT1. In contrast, an I Kr chemical activator of primary effects in slowing channel deactivation was not effective in modulating action potential durations. Our studies provide both the theoretical basis and experimental support for compensatory normalization of action potential duration by a pharmacological agent.
The transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated nonselective cation channel linked to human cardiac diseases. The human mutation K914R within TRPM4's S4-S5 linker was identified in patients with atrioventricular block. During UV-flash-mediated Ca2+ transients, TRPM4K914R generated a threefold augmented membrane current concomitant with 2 to 3-fold slowed down activation and deactivation kinetics resulting in excessive membrane currents during human cardiac action potentials. Mutagenesis of K914 paired with molecular modeling suggested the importance of the nanoscopic interface between the S4-S5 linker, the MHR4-, and TRP-domain as a major determinant for TRPM4's behavior. Rational mutagenesis of an interacting amino acid (R1062Q) in the TRP domain was able to offset K914R`s gain-of-function by zipping and unzipping of this nanoscopic interface. In conclusion, repulsion and attraction between the amino acids at positions 914 and 1062 alters the flexibility of the nanoscopic interface suggesting a zipping and unzipping mechanism that modulates TRPM4's functions. Pharmacological modulation of this intramolecular mechanism might represent a novel therapeutic strategy for the management of TRPM4-mediated cardiac diseases.
Systemic corticosteroids have been used to treat active inflammatory bowel disease for over 50 years by virtue of their unquestionable efficacy in inducing clinical remission rapidly in the vast majority of patients. Nevertheless, traditional corticosteroids are associated to a plethora of potentially serious side effects due to their systemic metabolism; for this reason, interest has lately been growing in newer steroid compounds characterized by a high topical anti-inflammatory activity and a low systemic bioavailability. These compounds, namely budesonide and beclomethasone dipropionate--regarding the treatment of inflammatory bowel disease--can be administered orally and thanks to sophisticated delivery systems are conveyed specifically to the inflamed gut mucosa where they exert their anti-inflammatory action. After intestinal absorption, these drugs are promptly and efficiently inactivated by the liver, so that only inactive molecules reach the systemic circulation. This review revises the main clinical trials, meta-analyses and observational studies conducted on traditional and newer steroids, and critically interprets the main results achieved by these studies.
During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1(+) precursors. Embryos deficient for Nkx2-5 in the Isl1(+) lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.
Myosin-10, also known as non-muscle myosin IIB, is a cytoskeletal protein implicated in cardiac development and disease. In humans, it is encoded by the MYH10 gene. Using CRISPR/Cas9 gene editing technology, we generated two MYH10 knockout human iPSC lines – one heterozygous (MRli003-A-1) and one homozygous (MRli003-A-2) – by introducing a frameshift deletion in exon 2. We then verified that both lines had maintained pluripotency, parental cell morphology, trilineage differentiation potential and a normal karyotype.
By combining single-channel and whole-cell patch-clamp recordings, we have established the sensitivity to ω-agatoxin IVA and ω-conotoxin MVIIC (SNX-230) of G1, G2, and G3, the three novel non-L-, non-N-type Ca 2+ channels characterized previously in rat cerebellar granule cells. G1 channels were blocked irreversibly by both ω-conotoxin MVIIC and low doses of ω-agatoxin IVA (saturation at 50 n m ). Thus, according to pharmacological criteria, G1 channels must be classified as P-type Ca 2+ channels. Being slowly inactivating during depolarizing pulses and completely inactivated at voltages in which steady-state inactivation of P-type channels in Purkinje cells is negligible, G1 represents a novel P subtype. Neither G2 nor G3 was blocked irreversibly by ω-conotoxin MVIIC, and therefore both are R-type Ca 2+ channels. G2 and G3 have some biophysical properties similar to those of low-voltage-activated (LVA) Ca 2+ channels (e.g., voltage range for steady-state inactivation, V 1/2 = −90 mV), some properties similar to those of high-voltage-activated (HVA) Ca 2+ channels (e.g., high sensitivity to Cd 2+ block), and other properties intermediate between those of LVA and HVA Ca 2+ channels, with LVA properties prevailing in G2 and HVA properties prevailing in G3. The R-type whole-cell current was inhibited by Ni 2+ with a biphasic dose–response curve (IC 50 : 4 and 153 μ m ), suggesting that G2 and G3 may have a different sensitivity to Ni 2+ block. Our results uncover functional diversity of both native P-type and R-type Ca 2+ channels and show that R subtypes with distinct biophysical properties are coexpressed in rat cerebellar granule cells.
Ectopic expression of defined sets of genetic factors can reprogram somatic cells to create induced pluripotent stem (iPS) cells. The capacity to direct human iPS cells to specific differentiated lineages and to their progenitor populations can be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. During mouse cardiogenesis, the major lineages of the mature heart, cardiomyocytes, smooth muscle cells, and endothelial cells arise from a common, multipotent cardiovascular progenitor expressing the transcription factors Isl1 and Nkx2.5. Here we show, using genetic fate-mapping, that Isl1+ multipotent cardiovascular progenitors can be generated from mouse iPS cells and spontaneously differentiate in all 3 cardiovascular lineages in vivo without teratoma. Moreover, we report the identification of human iPS-derived ISL1+ progenitors with similar developmental potential. These results support the possibility to use patient-specific iPS-generated cardiovascular progenitors as a model to elucidate the pathogenesis of congenital and acquired forms of heart diseases.—Moretti, A., Bellin, M., Jung, C. B., Thies, T.-M., Takashima, Y., Bernshausen, A., Schiemann, M., Fischer, S., Moosmang, S., Smith, A. G., Lam, J. T., Laugwitz, K.-L. Mouse and human induced pluripotent stem cells as a source for multipotent Isl1+ cardiovascular progenitors. FASEB J. 24, 700–711 (2010). www.fasebj.org
