The permeability of the glomerular filtration barrier (GFB) is mainly regulated by podocytes and their foot processes. Protein kinase G type Iα (PKGIα) and adenosine monophosphate-dependent kinase (AMPK) affect the contractile apparatus of podocytes and influence the permeability of the GFB. Therefore, we studied the interplay between PKGIα and AMPK in cultured rat podocytes. The glomerular permeability to albumin and transmembrane FITC-albumin flux decreased in the presence of AMPK activators and increased in the presence of PKG activators. The knockdown of PKGIα or AMPK with small-interfering RNA (siRNA) revealed a mutual interaction between PKGIα and AMPK and influenced podocyte permeability to albumin. Moreover, PKGIα siRNA activated the AMPK-dependent signaling pathway. AMPKα2 siRNA increased basal levels of phosphorylated myosin phosphate target subunit 1 and decreased the phosphorylation of myosin light chain 2. Podocytes that were treated with AMPK or PKG activators were characterized by the different organization of actin filaments within the cell. Our findings suggest that mutual interactions between PKGIα and AMPKα2 regulate the contractile apparatus and permeability of the podocyte monolayer to albumin. Understanding this newly identified molecular mechanism in podocytes provides further insights into the pathogenesis of glomerular disease and novel therapeutic targets for glomerulopathies.

Smooth muscle cells (SMCs) execute important physiological functions in numerous vital organ systems, including the vascular, gastrointestinal, respiratory, and urogenital tracts. SMC differ morphologically and functionally at these different anatomical locations, but the molecular underpinnings of the differences remain poorly understood. Here, using deep single-cell RNA sequencing combined with in situ gene and protein expression analysis in four murine organs-heart, aorta, lung, and colon-we identify a molecular basis for high-level differences among vascular, visceral, and airway SMC, as well as more subtle differences between, for example, SMC in elastic and muscular arteries and zonation of elastic artery SMC along the direction of blood flow. Arterial SMC exhibit extensive organotypic heterogeneity, whereas venous SMC are similar across organs. We further identify a specific SMC subtype within the pulmonary vasculature. This comparative SMC cross-organ resource offers insight into SMC subtypes and their specific functions.

Intermediate filaments (IFs), composed primarily by desmin and keratins, link the myofibrils to each other, to intracellular organelles, and to the sarcolemma. There they may play an important role in transfer of contractile force from the Z-disks and M-lines of neighboring myofibrils to costameres at the membrane, across the membrane to the extracellular matrix, and ultimately to the tendon (\"lateral force transmission\"). We measured the elasticity of the sarcolemma and the connections it makes at costameres with the underlying contractile apparatus of individual fast twitch muscle fibers of desmin-null mice. By positioning a suction pipet to the surface of the sarcolemma and applying increasing pressure, we determined the pressure at which the sarcolemma separated from nearby sarcomeres, Pseparation, and the pressure at which the isolated sarcolemma burst, Pbursting. We also examined the time required for the intact sarcolemma-costamere-sarcomere complex to reach equilibrium at lower pressures. All measurements showed the desmin-null fibers to have slower equilibrium times and lower Pseparation and Pbursting than controls, suggesting that the sarcolemma and its costameric links to nearby contractile structures were weaker in the absence of desmin. Comparisons to earlier values determined for muscles lacking dystrophin or synemin suggest that the desmin-null phenotype is more stable than the former and less stable than the latter. Our results are consistent with the moderate myopathy seen in desmin-null muscles and support the idea that desmin contributes significantly to sarcolemmal stability and lateral force transmission.

In vitro studies have shown that alterations in redox state can cause a range of opposing effects on the properties of the contractile apparatus in skeletal muscle fibers. To test whether and how redox changes occurring in vivo affect the contractile properties, vastus lateralis muscle fibers from seven healthy young adults were examined at rest (PRE) and following (POST) high-intensity intermittent cycling exercise. Individual mechanically skinned muscle fibers were exposed to heavily buffered solutions at progressively higher free [Ca2+] to determine their force-Ca2+ relationship. Following acute exercise, Ca2+ sensitivity was significantly decreased in type I fibers (by 0.06 pCa unit) but not in type II fibers (0.01 pCa unit). Specific force decreased after the exercise in type II fibers (-18%) but was unchanged in type I fibers. Treatment with the reducing agent dithiothreitol (DTT) caused a small decrease in Ca2+-sensitivity in type II fibers at PRE (by ∼0.014 pCa units) and a significantly larger decrease at POST (∼0.035 pCa units), indicating that the exercise had increased S-glutathionylation of fast troponin I. DTT treatment also increased specific force (by ∼4%), but only at POST. In contrast, DTT treatment had no effect on either parameter in type I fibers at either PRE or POST. In type I fibers, the decreased Ca2+ sensitivity was not due to reversible oxidative changes and may have contributed to a decrease in power production during vigorous exercises. In type II fibers, exercise-induced redox changes help counter the decline in Ca2+-sensitivity while causing a small decline in maximum force.NEW & NOTEWORTHY This study identified important cellular changes occurring in human skeletal muscle fibers following high-intensity intermittent exercise: 1) a decrease in contractile apparatus Ca2+ sensitivity in type I but not type II fibers, 2) a decrease in specific force only in type II muscle fibers, and 3) a redox-dependent increase in Ca2+ sensitivity occurring only in type II fibers, which would help maintain muscle performance by countering the normal metabolite-induced decline in Ca2+ sensitivity.

Although the O-GlcNAcylation process was discovered in 1984, its potential role in the physiology and physiopathology of skeletal muscle only emerged 20 years later. An increasing number of publications strongly support a key role of O-GlcNAcylation in the modulation of important cellular processes which are essential for skeletal muscle functions. Indeed, over a thousand of O-GlcNAcylated proteins have been identified within skeletal muscle since 2004, which belong to various classes of proteins, including sarcomeric proteins. In this review, we focused on these myofibrillar proteins, including contractile and structural proteins. Because of the modification of motor and regulatory proteins, the regulatory myosin light chain (MLC2) is related to several reports that support a key role of O-GlcNAcylation in the fine modulation of calcium activation parameters of skeletal muscle fibres, depending on muscle phenotype and muscle work. In addition, another key function of O-GlcNAcylation has recently emerged in the regulation of organization and reorganization of the sarcomere. Altogether, this data support a key role of O-GlcNAcylation in the homeostasis of sarcomeric cytoskeleton, known to be disturbed in many related muscle disorders.

The mechanically skinned (or \"peeled\") skeletal muscle fiber technique is a highly versatile procedure that allows controlled examination of each of the steps in the excitation-contraction (EC)-coupling sequence in skeletal muscle fibers, starting with excitation/depolarization of the transverse tubular (T)-system through to Ca2+ release from sarcoplasmic reticulum (SR) and finally force development by the contractile apparatus. It can also show the overall response of the whole EC-coupling sequence together, such as in twitch and tetanic force responses. A major advantage over intact muscle fiber preparations is that it is possible to set and rapidly manipulate the \"intracellular\" conditions, allowing examination of the effects of key variables (e.g., intracellular pH, ATP levels, redox state, etc.) on each individual step in EC coupling. This Cores of Reproducibility in Physiology (CORP) article describes the rationale, procedures, and experimental details of the various ways in which the mechanically skinned fiber technique is used in our laboratory to examine the physiological mechanisms controlling Ca2+ release and contraction in skeletal muscle fibers and the aberrations and dysfunction occurring with exercise and disease.

Nitric oxide is generated in skeletal muscle with activity and decreases Ca2+ sensitivity of the contractile apparatus, putatively by S-nitrosylation of an unidentified protein. We investigated the mechanistic basis of this effect and its relationship to the oxidation-induced increase in Ca2+ sensitivity in mammalian fast-twitch (FT) fibers mediated by S-glutathionylation of Cys134 on fast troponin I (TnIf). Force-[Ca2+] characteristics of the contractile apparatus in mechanically skinned fibers were assessed by direct activation with heavily Ca2+-buffered solutions. Treatment with S-nitrosylating agents, S-nitrosoglutathione (GSNO) or S-nitroso-N-acetyl-penicillamine (SNAP), decreased pCa50 ( = -log10 [Ca2+] at half-maximal activation) by ~-0.07 pCa units in rat and human FT fibers without affecting maximum force, but had no effect on rat and human slow-twitch fibers or toad or chicken FT fibers, which all lack Cys134. The Ca2+ sensitivity decrease was 1) fully reversed with dithiothreitol or reduced glutathione, 2) at least partially reversed with ascorbate, indicative of involvement of S-nitrosylation, and 3) irreversibly blocked by low concentration of the alkylating agent, N-ethylmaleimide (NEM). The biotin-switch assay showed that both GSNO and SNAP treatments caused S-nitrosylation of TnIfS-glutathionylation pretreatment blocked the effects of S-nitrosylation on Ca2+ sensitivity, and vice-versa. S-nitrosylation pretreatment prevented NEM from irreversibly blocking S-glutathionylation of TnIf and its effects on Ca2+ sensitivity, and likewise S-glutathionylation pretreatment prevented NEM block of S-nitrosylation. Following substitution of TnIf into rat slow-twitch fibers, S-nitrosylation treatment caused decreased Ca2+ sensitivity. These findings demonstrate that S-nitrosylation and S-glutathionylation exert opposing effects on Ca2+ sensitivity in mammalian FT muscle fibers, mediated by competitive actions on Cys134 of TnIf.

Myocardial remodeling in response to chronic myocardial infarction (CMI) progresses through two phases, hypertrophic \"compensation\" and congestive \"decompensation.\" Nothing is known about the ability of uninfarcted myocardium to produce force, velocity, and power during these clinical phases, even though adaptation in these regions likely drives progression of compensation. We hypothesized that enhanced cross-bridge-level contractility underlies mechanical compensation and is controlled in part by changes in the phosphorylation states of myosin regulatory proteins. We induced CMI in rats by left anterior descending coronary artery ligation. We then measured mechanical performance in permeabilized ventricular trabecula taken distant from the infarct zone and assayed myosin regulatory protein phosphorylation in each individual trabecula. During full activation, the compensated myocardium produced twice as much power and 31% greater isometric force compared with noninfarcted controls. Isometric force during submaximal activations was raised >2.4-fold, while power was 2-fold greater. Electron and confocal microscopy demonstrated that these mechanical changes were not a result of increased density of contractile protein and therefore not an effect of tissue hypertrophy. Hence, sarcomere-level contractile adaptations are key determinants of enhanced trabecular mechanics and of the overall cardiac compensatory response. Phosphorylation of myosin regulatory light chain (RLC) increased and remained elevated post-MI, while phosphorylation of myosin binding protein-C (MyBP-C) was initially depressed but then increased as the hearts became decompensated. These sensitivities to CMI are in accordance with phosphorylation-dependent regulatory roles for RLC and MyBP-C in crossbridge function and with compensatory adaptation in force and power that we observed in post-CMI trabeculae.

Cardiomyocytes in culture undergo reversible rearrangement of their contractile apparatus with the conversion of typical myofibrils into the structures of non-muscle type and the loss of contractility. Along with these transformations, the cardiomyocytes gain the capacity to synthesize extracellular matrix. Here we show that during cultivation of rat neonatal cardiomyocytes, the inherent α-cardiac actin isoform is transiently replaced by α-smooth-muscle actin, whose expression is accompanied by transformation of myofibrils into stress-fiber-like structures. The following down-regulation of α-smooth muscle actin parallels restoration of myofibrillar system and correlates with the accumulation of extracellular collagen and laminin, initially missing from the cardiomyocytes culture.

BACKGROUND: Multiple bone morphogenetic protein (BMP) genes are expressed in the developing heart from the initiation to late-differentiation stages, and play pivotal roles in cardiovascular development. In this study, we investigated the requirement of BMP activity in heart development by transgenic over-expression of extracellular BMP antagonist Noggin.
RESULTS: Using Nkx2.5-Cre to drive lineage-restricted Noggin within cardiomyocyte progenitors, we show persistent Noggin arrests cardiac development at the linear heart stage. This is coupled with a significantly reduced cell proliferation rate, subsequent cardiomyocyte programmed cell death and reduction of downstream intracellular pSMAD1/5/8 expression. Noggin mutants exhibit reduced heartbeat which likely results in subsequent fully penetrant in utero lethality. Significantly, confocal and electron micrographic examination revealed considerably fewer contractile elements, as well as a lack of maturation of actin-myosin microfilaments. Molecular analysis demonstrated that ectopic Noggin-expressing regions in the early heart\'s pacemaker region, failed to express the potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (Hcn4), resulting in an overall decrease in Hcn4 levels.
CONCLUSIONS: Combined, our results reveal a novel role for BMP signaling in the progression of heart development from the tubular heart stage to the looped stage by means of regulation of proliferation and promotion of maturation of the in utero heart\'s contractile apparatus and pacemaker.