The Contractile Machines of the Heart.

This chapter will describe basic structural and functional features of the contractile apparatus of muscle cells of the heart, namely, cardiomyocytes and smooth muscle cells. Cardiomyocytes form the contractile myocardium of the heart, while smooth muscle cells form the contractile coronary vessels. Both muscle types have distinct properties and will be considered with respect to their cellular appearance (brick-like cross-striated versus spindle-like smooth), arrangement of contractile proteins (sarcomeric versus non-sarcomeric organization), calcium activation mechanisms (thin-filament versus thick-filament regulation), contractile features (fast and phasic versus slow and tonic), energy metabolism (high oxygen versus low oxygen demand), molecular motors (type II myosin isoenzymes with high adenosine diphosphate [ADP]-release rate versus myosin isoenzymes with low ADP-release rates), chemomechanical energy conversion (high adenosine triphosphate [ATP] consumption and short duty ratio versus low ATP consumption and high duty ratio of myosin II cross-bridges [XBs]), and excitation-contraction coupling (calcium-induced calcium release versus pharmacomechanical coupling). Part of the work has been published (Neuroscience - From Molecules to Behavior", Chap. 22, Galizia and Lledo eds 2013, Springer-Verlag; with kind permission from Springer Science + Business Media).

Age-related decline in murine heart and skeletal muscle performance is attenuated by reduced Ahnak1 expression.

BACKGROUND: Aging is associated with a progressive reduction in cellular function leading to poor health and loss of physical performance. Mitochondrial dysfunction is one of the hallmarks of aging; hence, interventions targeting mitochondrial dysfunction have the potential to provide preventive and therapeutic benefits to elderly individuals. Meta-analyses of age-related gene expression profiles showed that the expression of Ahnak1, a protein regulating several signal-transduction pathways including metabolic homeostasis, is increased with age, which is associated with low VO2MAX and poor muscle fitness. However, the role of Ahnak1 in the aging process remained unknown. Here, we investigated the age-related role of Ahnak1 in murine exercise capacity, mitochondrial function, and contractile function of cardiac and skeletal muscles. METHODS: We employed 15- to 16-month-old female and male Ahnak1-knockout (Ahnak1-KO) and wild-type (WT) mice and performed morphometric, biochemical, and bioenergetics assays to evaluate the effects of Ahnak1 on exercise capacity and mitochondrial morphology and function in cardiomyocytes and tibialis anterior (TA) muscle. A human left ventricular (LV) cardiomyocyte cell line (AC16) was used to investigate the direct role of Ahnak1 in cardiomyocytes. RESULTS: We found that the level of Ahnak1 protein is significantly up-regulated with age in the murine LV (1.9-fold) and TA (1.8-fold) tissues. The suppression of Ahnak1 was associated with improved exercise tolerance, as all aged adult Ahnak1-KO mice (100%) successfully completed the running programme, whereas approximately 31% male and 8% female WT mice could maintain the required running speed and distance. Transmission electron microscopic studies showed that LV and TA tissue specimens of aged adult Ahnak1-KO of both sexes have significantly more enlarged/elongated mitochondria and less small mitochondria compared with WT littermates (P < 0.01 and P < 0.001, respectively) at basal level. Further, we observed a shift in mitochondrial fission/fusion balance towards fusion in cardiomyocytes and TA muscle from aged adult Ahnak1-KO mice. The maximal and reserve respiratory capacities were significantly higher in cardiomyocytes from aged adult Ahnak1-KO mice compared with the WT counterparts (P < 0.05 and P < 0.01, respectively). Cardiomyocyte contractility and fatigue resistance of TA muscles were significantly increased in Ahnak1-KO mice of both sexes, compared with the WT groups. In vitro studies using AC16 cells have confirmed that the alteration of mitochondrial function is indeed a direct effect of Ahnak1. Finally, we presented Ahnak1 as a novel cardiac mitochondrial membrane-associated protein. CONCLUSIONS: Our data suggest that Ahnak1 is involved in age-related cardiac and skeletal muscle dysfunction and could therefore serve as a promising therapeutical target.

Protective Function of Ahnak1 in Vascular Healing after Wire Injury.

AIM: Vascular remodeling following injury substantially accounts for restenosis and adverse clinical outcomes. In this study, we investigated the role of the giant scaffold protein Ahnak1 in vascular healing after endothelial denudation of the murine femoral artery. METHODS: The spatiotemporal expression pattern of Ahnak1 and Ahnak2 was examined using specific antibodies and real-time quantitative PCR. Following wire-mediated endothelial injury of Ahnak1-deficient mice and wild-type (WT) littermates, the processes of vascular healing were analyzed. RESULTS: Ahnak1 and Ahnak2 showed a mutually exclusive vascular expression pattern, with Ahnak1 being expressed in the endothelium and Ahnak2 in the medial cells in naïve WT arteries. After injury, a marked increase of Ahnak1- and Ahnak2-positive cells at the lesion site became evident. Both proteins showed a strong upregulation in neointimal cells 14 days after injury. Ahnak1-deficient mice showed delayed vascular healing and dramatically impaired re-endothelialization that resulted in prolonged adverse vascular remodeling, when compared to the WT littermates. CONCLUSION: The large scaffold and adaptor proteins Ahnak1 and Ahnak2 exhibit differential expression patterns and functions in naïve and injured arteries. Ahnak1 plays a nonredundant protective role in vascular healing.

17ß-Estradiol-induced interaction of estrogen receptor α and human atrial essential myosin light chain modulates cardiac contractile function.

Chronic increased workload of the human heart causes ventricular hypertrophy, re-expression of the atrial essential myosin light chain (hALC-1), and improved contractile function. Although hALC-1 is an important positive inotropic regulator of the human heart, little is known about its regulation. Therefore, we investigated the role of the sex hormone 17ß-estradiol (E2) on hALC-1 gene expression, the underlying molecular mechanisms, and the impact of this regulatory process on cardiac contractile function. We showed that E2 attenuated hALC-1 expression in human atrial tissues of both sexes and in human ventricular AC16 cells. E2 induced the nuclear translocation of estrogen receptor alpha (ERα) and hALC-1 in AC16 cells, where they cooperatively regulate the transcriptional activity of hALC-1 gene promoter. E2-activated ERα required the estrogen response element (ERE) motif within the hALC-1 gene promoter to reduce its transcriptional activity (vehicle: 15.55 ± 4.80 vs. E2: 6.51 ± 3.69; ~2 fold). This inhibitory effect was potentiated in the presence of hALC-1 (vehicle: 11.13 ± 3.66 vs. E2: 2.18 ± 1.10; ~5 fold), and thus, hALC-1 acts as a co-repressor of ERα-mediated transcription. Yeast two-hybrid screening of a human heart cDNA library revealed that ERα interacts physically with hALC-1 in the presence of E2. This interaction was confirmed by Co-Immunoprecipitation and immunofluorescence in human atrium. As a further novel effect, we showed that chronic E2-treatment of adult mouse cardiomyocytes overexpressing hALC-1 resulted in reduced cell-shortening amplitude and twitching kinetics of these cells independent of Ca2+ activation levels. Together, our data showed that the expression of hALC-1 gene is, at least partly, regulated by E2/ERα, while hALC-1 acts as a co-repressor. The inotropic effect of hALC-1 overexpression in cardiomyocytes can be significantly repressed by E2.

Muscle RING-finger 2 and 3 maintain striated-muscle structure and function.

BACKGROUND: The Muscle-specific RING-finger (MuRF) protein family of E3 ubiquitin ligases is important for maintenance of muscular structure and function. MuRF proteins mediate adaptation of striated muscles to stress. MuRF2 and MuRF3 bind to microtubules and are implicated in sarcomere formation with noticeable functional redundancy. However, if this redundancy is important for muscle function in vivo is unknown. Our objective was to investigate cooperative function of MuRF2 and MuRF3 in the skeletal muscle and the heart in vivo. METHODS: MuRF2 and MuRF3 double knockout mice (DKO) were generated and phenotypically characterized. Skeletal muscle and the heart were investigated by morphological measurements, histological analyses, electron microscopy, immunoblotting, and real-time PCR. Isolated muscles were subjected to in vitro force measurements. Cardiac function was determined by echocardiography and working heart preparations. Function of cardiomyocytes was measured in vitro. Cell culture experiments and mass-spectrometry were used for mechanistic analyses. RESULTS: DKO mice showed a protein aggregate myopathy in skeletal muscle. Maximal force development was reduced in DKO soleus and extensor digitorum longus. Additionally, a fibre type shift towards slow/type I fibres occurred in DKO soleus and extensor digitorum longus. MuRF2 and MuRF3-deficient hearts showed decreased systolic and diastolic function. Further analyses revealed an increased expression of the myosin heavy chain isoform beta/slow and disturbed calcium handling as potential causes for the phenotype in DKO hearts. CONCLUSIONS: The redundant function of MuRF2 and MuRF3 is important for maintenance of skeletal muscle and cardiac structure and function in vivo.

Cardiomyocyte-specific overexpression of oestrogen receptor ß improves survival and cardiac function after myocardial infarction in female and male mice.

ERß (oestrogen receptor ß) activation has been shown to be cardioprotective, but the cell types and mechanisms involved are not understood. To investigate whether ERß restricted to cardiomyocytes contributes to the observed cardioprotection, we tested the effects of cardiomyocyte-specific ERß-OE (ERß overexpression) on survival, cardiac remodelling and function after MI (myocardial infarction) and studied the molecular pathways potentially involved. Female and male mice with cardiomyocyte-specific ERß-OE and WT (wild-type) littermates were subjected to chronic anterior coronary artery ligation or sham surgery. Two weeks after MI, ERß-OE mice showed improved survival (100% and 83% compared with 76% and 58% in WT females and males respectively). ERß-OE was associated with attenuated LV (left ventricular) dilatation, smaller increase in heart weight, less lung congestion at similar MI size, and improved systolic and diastolic function in both sexes. We identified two potential pathways for ERß-mediated myocardial protection. First, male and female ERß-OE mice had a lower reduction of SERCA2a (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2a) expression after MI, suggesting less reduction in diastolic Ca(2+)-reuptake into the sarcoplasmic reticulum post-MI. Secondly, male ERß-OE revealed attenuated cardiac fibrosis in the remote LV tissue and expression of fibrosis markers collagen I and III, periostin and miR-21. Cardiomyocyte-specific ERß-OE improved survival associated with reduced maladaptive remodelling, improved cardiac function and less heart failure development after MI in both sexes. These effects seem to be related, at least in part, to a better maintenance of Ca(2+) cycling in both sexes and a lower induction of cardiac fibrosis in males after MI.

Susceptibility of murine induced pluripotent stem cell-derived cardiomyocytes to hypoxia and nutrient deprivation.

INTRODUCTION: Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) may be suitable for myocardial repair. While their functional and structural properties have been extensively investigated, their response to ischemia-like conditions has not yet been clearly defined. METHODS: iPS-CMs were differentiated and enriched from murine induced pluripotent stem cells expressing enhanced green fluorescent protein (eGFP) and puromycin resistance genes under the control of an α-myosin heavy chain (α-MHC) promoter. iPS-CMs maturity and function were characterized by microscopy, real-time PCR, calcium transient recordings, electrophysiology, and mitochondrial function assays, and compared to those from neonatal murine cardiomyocytes. iPS-CMs as well as neonatal murine cardiomyocytes were exposed for 3 hours to hypoxia (1% O2) and glucose/serum deprivation, and viability, apoptosis markers, reactive oxygen species, mitochondrial membrane potential and intracellular stress signaling cascades were investigated. Then, the iPS-CMs response to mesenchymal stromal cell-conditioned medium was determined. RESULTS: iPS-CMs displayed key morphological and functional properties that were comparable to those of neonatal cardiomyocytes, but several parameters indicated an earlier iPS-CMs maturation stage. During hypoxia and glucose/serum deprivation, iPS-CMs exhibited a significantly higher proportion of poly-caspase-active, 7-aminoactinomycin D-positive and TUNEL-positive cells than neonatal cardiomyocytes. The average mitochondrial membrane potential was reduced in "ischemic" iPS-CMs but remained unchanged in neonatal cardiomyocytes; reactive oxygen species production was only increased in "ischemic" iPS-CMs, and oxidoreductase activity in iPS-CMs dropped more rapidly than in neonatal cardiomyocytes. In iPS-CMs, hypoxia and glucose/serum deprivation led to upregulation of Hsp70 transcripts and decreased STAT3 phosphorylation and total PKCε protein expression. Treatment with mesenchymal stromal cell-conditioned medium preserved oxidoreductase activity and restored pSTAT3 and PKCε levels. CONCLUSION: iPS-CMs appear to be particularly sensitive to hypoxia and nutrient deprivation. Counteracting the ischemic susceptibility of iPS-CMs with mesenchymal stromal cell-conditioned medium may help enhance their survival and efficacy in cell-based approaches for myocardial repair.

Cardiomyocyte-specific Estrogen Receptor Alpha Increases Angiogenesis, Lymphangiogenesis and Reduces Fibrosis in the Female Mouse Heart Post-Myocardial Infarction.

Experimental studies showed that 17ß-estradiol (E2) and activated Estrogen Receptors (ER) protect the heart from ischemic injury. However, the underlying molecular mechanisms are not well understood. To investigate the role of ER-alpha (ERα) in cardiomyocytes in the setting of myocardial ischemia, we generated transgenic mice with cardiomyocyte-specific overexpression of ERα (ERα-OE) and subjected them to Myocardial Infarction (MI). At the basal level, female and male ERα-OE mice showed increased Left Ventricular (LV) mass, LV volume and cardiomyocyte length. Two weeks after MI, LV volume was significantly increased and LV wall thickness decreased in female and male WT-mice and male ERα-OE, but not in female ERα-OE mice. ERα-OE enhanced expression of angiogenesis and lymphangiogenesis markers (Vegf, Lyve-1), and neovascularization in the peri-infarct area in both sexes. However, attenuated level of fibrosis and higher phosphorylation of JNK signaling pathway could be detected only in female ERα-OE after MI. In conclusion, our study indicates that ERα protects female mouse cardiomyocytes from the sequelae of ischemia through induction of neovascularization in a paracrine fashion and impaired fibrosis, which together may contribute to the attenuation of cardiac remodelling.

Molecular mechanism regulating myosin and cardiac functions by ELC.

The essential myosin light chain (ELC) is involved in modulation of force generation of myosin motors and cardiac contraction, while its mechanism of action remains elusive. We hypothesized that ELC could modulate myosin stiffness which subsequently determines its force production and cardiac contraction. Therefore, we generated heterologous transgenic mouse (TgM) strains with cardiomyocyte-specific expression of ELC with human ventricular ELC (hVLC-1; TgM(hVLC-1)) or E56G-mutated hVLC-1 (hVLC-1(E56G); TgM(E56G)). hVLC-1 or hVLC-1(E56G) expression in TgM was around 39% and 41%, respectively of total VLC-1. Laser trap and in vitro motility assays showed that stiffness and actin sliding velocity of myosin with hVLC-1 prepared from TgM(hVLC-1) (1.67 pN/nm and 2.3 µm/s, respectively) were significantly higher than myosin with hVLC-1(E56G) prepared from TgM(E56G) (1.25 pN/nm and 1.7 µm/s, respectively) or myosin with mouse VLC-1 (mVLC-1) prepared from C57/BL6 (1.41 pN/nm and 1.5 µm/s, respectively). Maximal left ventricular pressure development of isolated perfused hearts in vitro prepared from TgM(hVLC-1) (80.0 mmHg) were significantly higher than hearts from TgM(E56G) (66.2 mmHg) or C57/BL6 (59.3±3.9 mmHg). These findings show that ELCs decreased myosin stiffness, in vitro motility, and thereby cardiac functions in the order hVLC-1>hVLC-1(E56G)≈mVLC-1. They also suggest a molecular pathomechanism of hypertrophic cardiomyopathy caused by hVLC-1 mutations.

Distinct interactions between actin and essential myosin light chain isoforms.

Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein-protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with α-actin as well as α-actin with alanin-mutated ELC binding site (α-actin(ala3)) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of α-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to α-actin (KD=575 nM) was significantly (p<0.01) lower compared with the affinity of hVLC-1 to α-actin (KD=186 nM). The reduced affinity of hALC-1 to α-actin was mainly due to a significantly (p<0.01) lower association rate (kon: 1,018 M(-1)s(-1)) compared with kon of the hVLC-1/α-actin complex interaction (2,908 M(-1)s(-1)). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct α-actin interactions.

Amyloid-ß peptides activate α1-adrenergic cardiovascular receptors.

Alzheimer disease features amyloid-ß (Aß) peptide deposition in brain and blood vessels and is associated with hypertension. Aß peptide can cause vasoconstriction and endothelial dysfunction. We observed that Aß peptides exert a chronotropic effect in neonatal cardiomyocytes, similar to α1-adrenergic receptor autoantibodies that we described earlier. Recently, it was shown that α1-adrenergic receptor could impair blood-brain flow. We hypothesized that Aß peptides might elicit a signal transduction pathway in vascular cells, induced by α1-adrenergic receptor activation. Aß (25-35) and Aß (10-35) induced a positive chronotropic effect in the cardiac contraction assay (28.75±1.15 and 29.40±0.98 bpm), which was attenuated by α1-adrenergic receptor blockers (urapidil, 1.53±1.17 bpm; prazosin, 0.30±0.96 bpm). Both Aß peptides induced an intracellular calcium release in vascular smooth muscle cells. Chronotropic activity and calcium response elicited by Aß (25-35) were blocked with peptides corresponding to the first extracellular loop of the α1-adrenergic receptor. We observed an induction of extracellular-regulated kinase 1/2 phosphorylation by Aß (25-35) in Chinese hamster ovary cells overexpressing α1-adrenergic receptor, vascular smooth muscle cells, and cardiomyocytes. We generated an activation-state-sensitive α1-adrenergic receptor antibody and visualized activation of the α1-adrenergic receptor by Aß peptide. Aß (25-35) induced vasoconstriction of mouse aortic rings and in coronary arteries in Langendorff-perfused rat hearts that resulted in decreased coronary flow. Both effects could be reversed by α1-adrenergic receptor blockade. Our data are relevant to the association between Alzheimer disease and hypertension. They may explain impairment of vascular responses by Aß and could have therapeutic implications.

Ahnak1 interaction is affected by phosphorylation of Ser-296 on Cavß2.

Ahnak1 has been implicated in protein kinase A (PKA)-mediated control of cardiac L-type Ca(2+) channels (Cav1.2) through its interaction with the Cavß(2) regulatory channel subunit. Here we corroborate this functional linkage by immunocytochemistry on isolated cardiomyocytes showing co-localization of ahnak1 and Cavß(2) in the T-tubule system. In previous studies Cavß(2) attachment sites which impacted the channel's PKA regulation have been located to ahnak1's proximal C-terminus (ahnak1(4889-5535), ahnak1(5462-5535)). In this study, we mapped the ahnak1-interacting regions in Cavß(2) and investigated whether Cavß(2) phosphorylation affects its binding behavior. In vitro binding assays with Cavß(2) truncation mutants and ahnak1(4889-5535) revealed that the core region of Cavß(2) consisting of Src-homology 3 (SH3), HOOK, and guanylate kinase (GK) domains was important for ahnak1 interaction while the C- and N-terminal regions were dispensable. Furthermore, Ser-296 in the GK domain of Cavß(2) was identified as novel PKA phosphorylation site by mass spectrometry. Surface plasmon resonance (SPR) binding analysis showed that Ser-296 phosphorylation did not affect the high affinity interaction (K(D)≈35 nM) between Cavß(2) and the α(1C) I-II linker, but affected ahnak1 interaction in a complex manner. SPR experiments with ahnak1(5462-5535) revealed that PKA phosphorylation of Cavß(2) significantly increased the binding affinity and, in parallel, it reduced the binding capacity. Intriguingly, the phosphorylation mimic substitution Glu-296 fully reproduced both effects, increased the affinity by ≈2.4-fold and reduced the capacity by ≈60%. Our results are indicative for the release of a population of low affinity interaction sites following Cavß(2) phosphorylation on Ser-296. We propose that this phosphorylation event is one mechanism underlying ahnak1's modulator function on Cav1.2 channel activity.

Mutations of ventricular essential myosin light chain disturb myosin binding and sarcomeric sorting.

AIMS: We tested the hypothesis that mutations in the human ventricular essential myosin light chain (hVLC-1) that are associated with hypertrophic cardiomyopathy (HCM) affect protein structure, binding to the IQ1 motif of cardiac myosin heavy chain (MYH) and sarcomeric sorting in neonatal cardiomyocytes. METHODS AND RESULTS: We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties and protein-protein interactions of a recombinant head-rod fragment of rat cardiac ß-MYH (amino acids 664-915) with alanine-mutated IQ2 domain (rß-MYH(664-915)IQ2(ala4)) and normal or five mutated (M149V, E143K, A57G, E56G, R154H) hVLC-1 forms. Double epitope-tagging competition was used to monitor the intracellular localization of exogenously introduced normal and E56G-mutated (hVLC-1(E56G)) hVLC-1 constructs in neonatal rat cardiomyocytes. Fluorescence lifetime imaging microscopy was applied to map the microenvironment of normal and E56G-mutated hVLC-1 in permeabilized muscle fibres. Affinity of M149V, E143K, A57G, and R154H mutated hVLC-1/rß-MYH(664-915)IQ2(ala4) complexes was significantly lower compared with the normal hVLC-1/rß-MYH(664-915)IQ2(ala4) complex interaction. In particular, the E56G mutation induced an ∼30-fold lower MYH affinity. Sorting specificity of E56G-mutated hVLC-1 was negligible compared with normal hVLC-1. Fluorescence lifetime of fibres replaced with hVLC-1(E56G) increased significantly compared with hVLC-1-replaced fibres. CONCLUSION: Disturbed myosin binding of mutated hVLC-1 may provide a pathomechanism for the development of HCM.

Ahnak1 abnormally localizes in muscular dystrophies and contributes to muscle vesicle release.

Ahnak1 is a giant, ubiquitously expressed, plasma membrane support protein whose function in skeletal muscle is largely unknown. Therefore, we investigated whether ahnak would be influenced by alterations of the sarcolemma exemplified by dysferlin mutations known to render the sarcolemma vulnerable or by mutations in calpain3, a protease known to cleave ahnak. Human muscle biopsy specimens obtained from patients with limb girdle muscular dystrophy (LGMD) caused by mutations in dysferlin (LGMD2B) and calpain3 (LGMD2A) were investigated for ahnak expression and localization. We found that ahnak1 has lost its sarcolemmal localization in LGMD2B but not in LGMD2A. Instead ahnak1 appeared in muscle connective tissue surrounding the extracellular site of the muscle fiber in both muscular dystrophies. The entire giant ahnak1 molecule was present outside the muscle fiber and did only partially colocalize with CD45-positive immune cell infiltration and the extracelluar matrix proteins fibronectin and collagenVI. Further, vesicles shedded in response to Ca(2+) by primary human myotubes were purified and their protein content was analysed. Ahnak1 was prominently present in these vesicles. Electron microscopy revealed a homogenous population of vesicles with a diameter of about 150 nm. This is the first study demonstrating vesicle release from human myotubes that may be one mechanism underlying abnormally localized ahnak1. Taken together, our results define ahnak1 in muscle connective tissue as a novel feature of two genetically distinct muscular dystrophies that might contribute to disease pathology.

Ahnak1 is a tuneable modulator of cardiac Ca(v)1.2 calcium channel activity.

Ahnak1 has been implicated in the beta-adrenergic regulation of the cardiac L-type Ca(2+) channel current (I (CaL)) by its binding to the regulatory Cavß(2) subunit. In this study, we addressed the question whether ahnak1/Cavß(2) interactions are essential or redundant for beta-adrenergic stimulation of I (CaL). Three naturally occurring ahnak1 variants (V5075 M, G5242R, and T5796 M) identified by genetic screening of cardiomyopathy patients did essentially not influence the in vitro Cavß(2) interaction as assessed by recombinant proteins. But, we observed a robust increase in Cavß(2) binding by mutating Ala at position 4984 to Pro which creates a PxxP consensus motif in the ahnak1 protein fragment. Surface plasmon resonance measurements revealed that this mutation introduced an additional Cavß(2) binding site. The functionality of A4984P was supported by the specific action of the Pro-containing ahnak1-derived peptide (P4984) in beta-adrenergic regulation of I (CaL). Patch clamp recordings on cardiomyocytes showed that intracellular perfusion of P4984 markedly reduced I (CaL) response to the beta-adrenergic agonist, isoprenaline, while the Ala-containing counterpart failed to affect I (CaL). Interestingly, I (CaL) of ahnak1-deficient cardiomyocytes was not affected by peptide application. Moreover, I (CaL) of ahnak1-deficient cardiomyocytes showed intact beta-adrenergic responsiveness. Similarly isolated ahnak1-deficient mouse hearts responded normally to adrenergic challenge. Our results indicate that ahnak1 is not essential for beta-adrenergic up-regulation of I (CaL) and cardiac contractility in mice. But, tuning ahnak1/Cavß(2) interaction provides a tool for modulating the beta-adrenergic response of I (CaL).

Spinophilin is required for normal morphology, Ca(2+) homeostasis and contraction but dispensable for ß-adrenergic stimulation of adult cardiomyocytes.

Spinophilin (SPN) is a ubiquitously expressed scaffolding protein that interacts through several binding modules with a variety of target proteins. Thus, SPN bundles F-actin, targets protein phosphatase 1 to the ryanodine receptor, and targets regulators of G-protein signaling to G-protein coupled receptors in cardiomyocytes. In this work we studied the role of SPN on cardiomyocyte morphology, function, and ß-adrenergic responsiveness using a homozygous SPN knock-out mouse model (SPN-/-). We show that spinophilin deficiency significantly (1) reduced cardiomyocyte length, (2) increases both Ca(2+) amplitude and maximal rate of Ca(2+) rise during systole, and (3) decreased shortening amplitude and maximal rate of shortening, while (4) ß-adrenergic stimulation remained intact. Our data suggest that spinophilin is an upstream regulator required for normal growth and excitation-contraction coupling, but is dispensable for ß-adrenergic stimulation of adult cardiomyocytes.

Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides.

In addition to endocytosis-mediated cellular uptake, hydrophilic cell-penetrating peptides are able to traverse biological membranes in a non-endocytic mode termed transduction, resulting in immediate bioavailability. Here we analysed structural requirements for the non-endocytic uptake mode of arginine-rich cell-penetrating peptides, by a combination of live-cell microscopy, molecular dynamics simulations and analytical ultracentrifugation. We demonstrate that the transduction efficiency of arginine-rich peptides increases with higher peptide structural rigidity. Consequently, cyclic arginine-rich cell-penetrating peptides showed enhanced cellular uptake kinetics relative to their linear and more flexible counterpart. We propose that guanidinium groups are forced into maximally distant positions by cyclization. This orientation increases membrane contacts leading to enhanced cell penetration.

BMS309403 directly suppresses cardiac contractile function.

BMS309403, a substance used as an inhibitor of adipocyte fatty acid-binding protein, has been suggested as a new therapeutic agent for treating type 2 diabetes mellitus and atherosclerosis; however, little is known about its possible side effects. The present study investigates the effects of BMS309403 on the cardiovascular system. We used isolated perfused heart preparations and single cardiomyocytes from adult rats for contractile analysis. The Ca(2+) sensitivity of the myofilaments was investigated by using porcine cardiac skinned muscle fibers. BMS309403 induced a negative effect on the contractility of isolated perfused hearts leading to heart arrest without interfering in the electrocardiographic activity, suggesting electromechanical dissociation. Experiments with isolated cardiomyocytes showed that BMS309403 had a direct biphasic inhibitory effect on cardiomyocyte contraction, at higher concentrations by attenuating Ca(2+) levels. This negative inotropic effect does not result from a direct effect on the myofilaments. BMS309403 has an acute cardiac depressant effect in vitro. The potential therapeutic applicability of this compound requires additional consideration.

Human essential myosin light chain isoforms revealed distinct myosin binding, sarcomeric sorting, and inotropic activity.

AIMS: In this paper, we tested the hypothesis that different binding affinities of the C-terminus of human cardiac alkali (essential) myosin light chain (A1) isoforms to the IQ1 motif of the myosin lever arm provide a molecular basis for distinct sarcomeric sorting and inotropic activity. METHODS AND RESULTS: We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties, secondary structures, and protein-protein interactions of a recombinant head-rod fragments of rat cardiac ß-myosin heavy chain aa664-915 with alanine-mutated IQ2 domain (rß-MYH(664-915)IQ(ala4)) and A1 isoforms [human atrial (hALC1) and human ventricular (hVLC-1) light chains]. Double epitope-tagging competition was used to monitor the intracellular localization of exogenously introduced hALC-1 and hVLC-1 constructs in neonatal rat cardiomyocytes. Contractile functions of A1 isoforms were investigated by monitoring shortening and intracellular-free Ca(2+) (Fura-2) of adult rat cardiomyocytes infected with adenoviral (Ad) vectors using hALC-1 or ß-galactosidase as expression cassettes. hALC-1 bound more strongly (greater than three-fold lower K(D)) to rß-MYH(664-915) than did hVLC-1. Sorting specificity of A1 isoforms to sarcomeres of cardiomyocytes rose in the order hVLC-1 to hALC-1. Replacement of endogenous VLC-1 by hALC-1 in adult rat cardiomyocytes increased contractility while the systolic Ca(2+) signal remained unchanged. CONCLUSION: Intense myosin binding of hALC-1 provides a mechanism for preferential sarcomeric sorting and Ca(2+)-independent positive inotropic activity.