Nina D. Ullrich

Reciprocal amplification of ROS and Ca2+ signals in stressed mdx dystrophic skeletal muscle fibers

Muscular dystrophies are among the most severe inherited muscle diseases. The genetic defect is a mutation in the gene for dystrophin, a cytoskeletal protein which protects muscle cells from mechanical damage. Mechanical stress, applied as osmotic shock, elicits an abnormal surge of Ca2+ spark-like events in skeletal muscle fibers from dystrophin deficient (mdx) mice. Previous studies suggested a link between changes in the intracellular redox environment and appearance of Ca2+ sparks in normal mammalian skeletal muscle. Here, we tested whether the exaggerated Ca2+ responses in mdx fibers are related to oxidative stress. Localized intracellular and mitochondrial Ca2+ transients, as well as ROS production, were assessed with confocal microscopy. The rate of basal cellular but not mitochondrial ROS generation was significantly higher in mdx cells. This difference was abolished by pre-incubation of mdx fibers with an inhibitor of NAD(P)H oxidase. In addition, immunoblotting showed a significantly stronger expression of NAD(P)H oxidase in mdx muscle, suggesting a major contribution of this enzyme to oxidative stress in mdx fibers. Osmotic shock produced an abnormal and persistent Ca2+ spark activity, which was suppressed by ROS-reducing agents and by inhibitors of NAD(P)H oxidase. These Ca2+ signals resulted in mitochondrial Ca2+ accumulation in mdx fibers and an additional boost in cellular and mitochondrial ROS production. Taken together, our results indicate that the excessive ROS production and the simultaneous activation of abnormal Ca2+ signals amplify each other, finally culminating in a vicious cycle of damaging events, which may contribute to the abnormal stress sensitivity in dystrophic skeletal muscle.

Posttranslational modifications of cardiac ryanodine receptors: Ca2+ signaling and EC-coupling

In cardiac muscle, a number of posttranslational protein modifications can alter the function of the Ca(2+) release channel of the sarcoplasmic reticulum (SR), also known as the ryanodine receptor (RyR). During every heartbeat RyRs are activated by the Ca(2+)-induced Ca(2+) release mechanism and contribute a large fraction of the Ca(2+) required for contraction. Some of the posttranslational modifications of the RyR are known to affect its gating and Ca(2+) sensitivity. Presently, research in a number of laboratories is focused on RyR phosphorylation, both by PKA and CaMKII, or on RyR modifications caused by reactive oxygen and nitrogen species (ROS/RNS). Both classes of posttranslational modifications are thought to play important roles in the physiological regulation of channel activity, but are also known to provoke abnormal alterations during various diseases. Only recently it was realized that several types of posttranslational modifications are tightly connected and form synergistic (or antagonistic) feed-back loops resulting in additive and potentially detrimental downstream effects. This review summarizes recent findings on such posttranslational modifications, attempts to bridge molecular with cellular findings, and opens a perspective for future work trying to understand the ramifications of crosstalk in these multiple signaling pathways. Clarifying these complex interactions will be important in the development of novel therapeutic approaches, since this may form the foundation for the implementation of multi-pronged treatment regimes in the future. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

PKA phosphorylation of cardiac ryanodine receptor modulates SR luminal Ca2+ sensitivity

During physical exercise and stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor activation, resulting in protein kinase A (PKA)-mediated phosphorylation of the cardiac ryanodine receptor, RyR2, at Ser2808. Hyperphosphorylation of RyR2-S2808 has been proposed as a mechanism contributing to arrhythmogenesis and heart failure. However, the role of RyR2 phosphorylation during β-adrenergic stimulation remains controversial. We examined the contribution of RyR2-S2808 phosphorylation to altered excitation-contraction coupling and Ca(2+) signaling using an experimental approach at the interface of molecular and cellular levels and a transgenic mouse with ablation of the RyR2-S2808 phosphorylation site (RyR2-S2808A). Experimentally challenging the communication between L-type Ca(2+) channels and RyR2 led to a spatiotemporal de-synchronization of RyR2 openings, as visualized using confocal Ca(2+) imaging. β-Adrenergic stimulation re-synchronized RyR2s, but less efficiently in RyR2-S2808A than in control cardiomyocytes, as indicated by comprehensive analysis of RyR2 activation. In addition, spontaneous Ca(2+) waves in RyR2-S2808A myocytes showed significantly slowed propagation and complete absence of acceleration during β-adrenergic stress, unlike wild type cells. Single channel recordings revealed an attenuation of luminal Ca(2+) sensitivity in RyR2-S2808A channels upon addition of PKA. This suggests that phosphorylation of RyR2-S2808 may be involved in RyR2 modulation by luminal (intra-SR) Ca(2+) ([Ca(2+)](SR)). We show here by three independent experimental approaches that PKA-dependent RyR2-S2808 phosphorylation plays significant functional roles at the subcellular level, namely, Ca(2+) release synchronization, Ca(2+) wave propagation and functional adaptation of RyR2 to variable [Ca(2+)](SR). These results indicate a direct mechanistic link between RyR2 phosphorylation and SR luminal Ca(2+) sensing.

Alterations of excitation-contraction coupling and excitation coupled Ca(2+) entry in human myotubes carrying CAV3 mutations linked to rippling muscle

Rippling muscle disease is caused by mutations in the gene encoding caveolin‐3 (CAV3), the muscle‐specific isoform of the scaffolding protein caveolin, a protein involved in the formation of caveolae. In healthy muscle, caveolin‐3 is responsible for the formation of caveolae, which are highly organized sarcolemmal clusters influencing early muscle differentiation, signalling and Ca2+ homeostasis. In the present study we examined Ca2+ homeostasis and excitation–contraction (E‐C) coupling in cultured myotubes derived from two patients with Rippling muscle disease with severe reduction in caveolin‐3 expression; one patient harboured the heterozygous c.84C>A mutation while the other patient harbored a homozygous splice‐site mutation (c.102+ 2T>C) affecting the splice donor site of intron 1 of the CAV3 gene. Our results show that cells from control and rippling muscle disease patients had similar resting [Ca2+]i and 4‐chloro‐m‐cresol‐induced Ca2+ release but reduced KCl‐induced Ca2+ influx. Detailed analysis of the voltage‐dependence of Ca2+ transients revealed a significant shift of Ca2+ release activation to higher depolarization levels in CAV3 mutated cells. High resolution immunofluorescence analysis by Total Internal Fluorescence microscopy supports the hypothesis that loss of caveolin‐3 leads to microscopic disarrays in the colocalization of the voltage‐sensing dihydropyridine receptor and the ryanodine receptor, thereby reducing the efficiency of excitation–contraction coupling. Hum Mutat 32:309–317, 2011.

Establishment of a human skeletal muscle-derived cell line: biochemical, cellular and electrophysiological characterization

Excitation-contraction coupling is the physiological mechanism occurring in muscle cells whereby an electrical signal sensed by the dihydropyridine receptor located on the transverse tubules is transformed into a chemical gradient (Ca2+ increase) by activation of the ryanodine receptor located on the sarcoplasmic reticulum membrane. In the present study, we characterized for the first time the excitation-contraction coupling machinery of an immortalized human skeletal muscle cell line. Intracellular Ca2+ measurements showed a normal response to pharmacological activation of the ryanodine receptor, whereas 3D-SIM (super-resolution structured illumination microscopy) revealed a low level of structural organization of ryanodine receptors and dihydropyridine receptors. Interestingly, the expression levels of several transcripts of proteins involved in Ca2+ homoeostasis and differentiation indicate that the cell line has a phenotype closer to that of slow-twitch than fast-twitch muscles. These results point to the potential application of such human muscle-derived cell lines to the study of neuromuscular disorders; in addition, they may serve as a platform for the development of therapeutic strategies aimed at correcting defects in Ca2+ homoeostasis due to mutations in genes involved in Ca2+ regulation.

‘Eventless’ InsP3‐dependent SR‐Ca2+ release affecting atrial Ca2+ sparks

•  Inositol 1,4,5‐trisphosphate receptors (InsP3Rs) are functionally expressed in cardiac myocytes. •  The influence of inositol 1,4,5‐trisphosphate‐induced sarcoplasmic reticulum (SR)‐Ca2+release (IP3ICR) on atrial excitation‐contraction coupling (ECC) under physiological and pathophysiological conditions remains elusive. •  The present study focuses on local IP3ICR and its functional consequences for ryanodine receptor (RyR) activity and subsequent Ca2+‐induced Ca2+ release in atrial myocytes. •  Here we show significant SR‐Ca2+ flux, but eventless SR‐Ca2+ release through InsP3Rs. •  We suggest a new mechanism based on eventless and highly efficient InsP3‐dependent SR‐Ca2+ flux as a crucial mechanism of functional cross‐talk between InsP3Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.

Eventless InsP 3 -dependent SR-Ca 2+ Release Affecting Atrial Ca 2+ Sparks

•  Inositol 1,4,5‐trisphosphate receptors (InsP3Rs) are functionally expressed in cardiac myocytes. •  The influence of inositol 1,4,5‐trisphosphate‐induced sarcoplasmic reticulum (SR)‐Ca2+release (IP3ICR) on atrial excitation‐contraction coupling (ECC) under physiological and pathophysiological conditions remains elusive. •  The present study focuses on local IP3ICR and its functional consequences for ryanodine receptor (RyR) activity and subsequent Ca2+‐induced Ca2+ release in atrial myocytes. •  Here we show significant SR‐Ca2+ flux, but eventless SR‐Ca2+ release through InsP3Rs. •  We suggest a new mechanism based on eventless and highly efficient InsP3‐dependent SR‐Ca2+ flux as a crucial mechanism of functional cross‐talk between InsP3Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.

Functional Characterization and Comparison of Intercellular Communication in Stem Cell‐Derived Cardiomyocytes

One novel treatment strategy for the diseased heart focuses on the use of pluripotent stem cell‐derived cardiomyocytes (SC‐CMs) to overcome the hearts innate deficiency for self‐repair. However, targeted application of SC‐CMs requires in‐depth characterization of their true cardiogenic potential in terms of excitability and intercellular coupling at cellular level and in multicellular preparations. In this study, we elucidated the electrical characteristics of single SC‐CMs and intercellular coupling quality of cell pairs, and concomitantly compared them with well‐characterized murine native neonatal and immortalized HL‐1 cardiomyocytes. Firstly, we investigated the electrical properties and Ca2+ signaling mechanisms specific to cardiac contraction in single SC‐CMs. Despite heterogeneity of the new cardiac cell population, their electrophysiological activity and Ca2+ handling were similar to native cells. Secondly, we investigated the capability of paired SC‐CMs to form an adequate subunit of a functional syncytium and analyzed gap junctions and signal transmission by dye transfer in cell pairs. We discovered significantly diminished coupling in SC‐CMs compared with native cells, which could not be enhanced by a coculture approach combining SC‐CMs and primary CMs. Moreover, quantitative and structural analysis of gap junctions presented significantly reduced connexin expression levels compared with native CMs. Strong dependence of intercellular coupling on gap junction density was further confirmed by computational simulations. These novel findings demonstrate that despite the cardiogenic electrophysiological profile, SC‐CMs present significant limitations in intercellular communication. Inadequate coupling may severely impair functional integration and signal transmission, which needs to be carefully considered for the prospective use of SC‐CMs in cardiac repair. Stem Cells 2015;33:2208–2218

Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

Functional characterization of orbicularis oculi and extraocular muscles

Facial muscles are skeletal muscles that control facial expression. Sekulic-Jablanovic et al. characterize orbicularis oculi and extraocular muscles and find divergence in the expression of key molecules for muscle function between facial, extraocular, and quadriceps muscles.