Reyes, Santiago

Significance Skeletal muscle is increasingly recognized as a secretory organ. Revealing the identity and function of myokines can improve our understanding of skeletal muscle function under sedentary or exercise conditions, as well as its coordination with other organs, tissues, and overall body metabolism. This study identifies musclin as an exercise-responsive myokine critical for skeletal muscle adaptation to physical activity. We develop a musclin-encoding gene ( Ostn ) knockout mouse, which allows us to determine a previously unrecognized physiologic function of musclin in regulation of skeletal muscle mitochondrial biogenesis and physical endurance. The demonstrated molecular mechanism for musclin-dependent skeletal muscle adaptation to exercise also transforms the perspective on natriuretic peptide signaling, particularly as it relates to physical activity and exercise-induced remodeling in different tissues.

Background—Nuclear reprogramming inculcates pluripotent capacity by which de novo tissue differentiation is enabled. Yet, introduction of ectopic reprogramming factors may desynchronize natural developmental schedules. This study aims to evaluate the effect of imposed transgene load on the cardiogenic competency of induced pluripotent stem (iPS) cells.Methods and Results—Targeted inclusion and exclusion of reprogramming transgenes (c-MYC,KLF4,OCT4, andSOX2) was achieved using a drug-inducible and removable cassette according to the piggyBac transposon/transposase system. Pulsed transgene overexpression, before iPS cell differentiation, hindered cardiogenic outcomes. Delayed in counterparts with maintained integrated transgenes, transgene removal enabled proficient differentiation of iPS cells into functional cardiac tissue. Transgene-free iPS cells generated reproducible beating activity with robust expression of cardiac α-actinin, connexin 43, myosin light chain 2a, α/β-myosin heavy chain, and troponin I. Although operational excitation–contraction coupling was demonstrable in the presence or absence of transgenes, factor-free derivatives exhibited an expedited maturing phenotype with canonical responsiveness to adrenergic stimulation.Conclusions—A disproportionate stemness load, caused by integrated transgenes, affects the cardiogenic competency of iPS cells. Offload of transgenes in engineered iPS cells ensures integrity of cardiac developmental programs, underscoring the value of nonintegrative nuclear reprogramming for derivation of competent cardiogenic regenerative biologics.

Background— Na V 1.5 is a mechanosensitive voltage-gated sodium-selective ion channel responsible for the depolarizing current and maintenance of the action potential plateau in the heart. Ranolazine is a Na V 1.5 antagonist with antianginal and antiarrhythmic properties. Methods and Results— Mechanosensitivity of Na V 1.5 was tested in voltage-clamped whole cells and cell-attached patches by bath flow and patch pressure, respectively. In whole cells, bath flow increased peak inward current in both murine ventricular cardiac myocytes (24±8%) and human embryonic kidney 293 cells heterologously expressing Na V 1.5 (18±3%). The flow-induced increases in peak current were blocked by ranolazine. In cell-attached patches from cardiac myocytes and Na V 1.5-expressing human embryonic kidney 293 cells, negative pressure increased Na V peak currents by 27±18% and 18±4% and hyperpolarized voltage dependence of activation by −11 mV and −10 mV, respectively. In human embryonic kidney 293 cells, negative pressure also increased the window current (250%) and increased late open channel events (250%). Ranolazine decreased pressure-induced shift in the voltage dependence (IC 50 54 μmol/L) and eliminated the pressure-induced increases in window current and late current event numbers. Block of Na V 1.5 mechanosensitivity by ranolazine was not due to the known binding site on DIVS6 (F1760). The effect of ranolazine on mechanosensitivity of Na V 1.5 was approximated by lidocaine. However, ionized ranolazine and charged lidocaine analog (QX-314) failed to block mechanosensitivity. Conclusions— Ranolazine effectively inhibits mechanosensitivity of Na V 1.5. The block of Na V 1.5 mechanosensitivity by ranolazine does not utilize the established binding site and may require bilayer partitioning. Ranolazine block of Na V 1.5 mechanosensitivity may be relevant in disorders of mechanoelectric dysfunction.

Rationale : Induced pluripotent stem cells (iPS) allow derivation of pluripotent progenitors from somatic sources. Originally, iPS were induced by a stemness-related gene set that included the c-MYC oncogene. Objective : Here, we determined from embryo to adult the cardiogenic proficiency of iPS programmed without c-MYC, a cardiogenicity-associated transcription factor. Methods and Results : Transgenic expression of 3 human stemness factors SOX2, OCT4, and KLF4 here reset murine fibroblasts to the pluripotent ground state. Transduction without c-MYC reversed cellular ultrastructure into a primitive archetype and induced stem cell markers generating 3-germ layers, all qualifiers of acquired pluripotency. Three-factor induced iPS (3F-iPS) clones reproducibly demonstrated cardiac differentiation properties characterized by vigorous beating activity of embryoid bodies and robust expression of cardiac Mef2c, α-actinin, connexin43, MLC2a, and troponin I. In vitro isolated iPS-derived cardiomyocytes demonstrated functional excitation-contraction coupling. Chimerism with 3F-iPS derived by morula-stage diploid aggregation was sustained during prenatal heart organogenesis and contributed in vivo to normal cardiac structure and overall performance in adult tumor-free offspring. Conclusions : Thus, 3F-iPS bioengineered without c-MYC achieve highest stringency criteria for bona fide cardiogenesis enabling reprogrammed fibroblasts to yield de novo heart tissue compatible with native counterpart throughout embryological development and into adulthood.

Allosteric regulation of heteromultimeric ATP-sensitive potassium (KATP) channels is unique among protein systems as it implies transmission of ligand-induced structural adaptation at the regulatory SUR subunit, a member of ATP-binding cassette ABCC family, to the distinct pore-forming K+ (Kir6.x) channel module. Cooperative interaction between nucleotide binding domains (NBDs) of SUR is a prerequisite for KATP channel gating, yet pathways of allosteric intersubunit communication remain uncertain. Here, we analyzed the role of the ED domain, a stretch of 15 negatively charged aspartate/glutamate amino acid residues (948–962) of the SUR2A isoform, in the regulation of cardiac KATP channels. Disruption of the ED domain impeded cooperative NBDs interaction and interrupted the regulation of KATP channel complexes by MgADP, potassium channel openers, and sulfonylurea drugs. Thus, the ED domain is a structural component of the allosteric pathway within the KATP channel complex integrating transduction of diverse nucleotide-dependent states in the regulatory SUR subunit to the open/closed states of the K+-conducting channel pore.

Exercise training elicits a metabolic and cardiovascular response that underlies fitness. The molecular mechanisms that orchestrate this adaptive response and secure the wide-ranging gains of a regimented exercise program are poorly understood. Formed through association of the Kir6.2 pore and the sulfonylurea receptor, the stress-responsive ATP-sensitive K+ channels (KATP channels), with their metabolic-sensing capability and broad tissue expression, are potential candidates for integrating the systemic adaptive response to repetitive exercise. Here, the responses of mice lacking functional Kir6.2-containing KATP channels (Kir6.2-KO) were compared with wild-type controls following a 28-day endurance swimming protocol. While chronic aquatic training resulted in lighter, leaner, and fitter wild-type animals, the Kir6.2-KO manifested less augmentation in exercise capacity and lacked metabolic improvement in body fat composition and glycemic handling with myocellular defects. Moreover, the repetitive stress of swimming unmasked a survival disadvantage in the Kir6.2-KO, associated with pathologic calcium-dependent structural damage in the heart and impaired cardiac performance. Thus, Kir6.2-containing KATP channel activity is required for attainment of the physiologic benefits of exercise training without injury.

Sepsis, the systemic inflammatory response to infection, imposes a high demand for bodily adaptation, with the cardiovascular response a key determinant of outcome. The homeostatic elements that secure cardiac tolerance in the setting of the sepsis syndrome are poorly understood. Here, in a model of acute septic shock induced by endotoxin challenge with Escherichia coli lipopolysaccharide (LPS), knockout of the KCNJ8 gene encoding the vascular Kir6.1 K ATP channel pore predisposed to an early and profound survival disadvantage. The exaggerated susceptibility provoked by disruption of this stress‐responsive sensor of cellular metabolism was linked to progressive deterioration in cardiac activity, ischemic myocardial damage, and contractile dysfunction. Deletion of KCNJ8 blunted the responsiveness of coronary vessels to cytokine‐ or metabolic‐mediated vasodilation necessary to support myocardial perfusion in the wild‐type (WT), creating a deficit in adaptive response in the Kir6.1 knockout. Application of a K ATP channel opener drug improved survival in the endotoxic WT but had no effect in the Kir6.1 knockout. Restoration of the dilatory capacity of coronary vessels was required to rescue the Kir6.1 knockout phenotype and reverse survival disadvantage in lethal endotoxemia. Thus, the Kir6.1containing K ATP channel, by coupling vasoreactivity with metabolic demand, provides a vital feedback element for cardiovascular tolerance in endotoxic shock.—Kane, G. C., Lam, C‐F., O'Cochlain, F., Hodgson, D. M., Reyes, S., Liu, X‐K., Miki, T., Seino, S., Katusic, Z. S., Terzic, A. Gene knockout of the KCNJ8encoded Kir6.1 KATP channel imparts fatal susceptibility to endotoxemia. FASEB J. 20, 2271–2280 (2006)