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).

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 VO and poor muscle fitness.

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.

17β-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 17β-estradiol (E2) on hALC-1 gene expression, the underlying molecular mechanisms, and the impact of this regulatory process on cardiac contractile function.

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.

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

ERβ (oestrogen receptor β) activation has been shown to be cardioprotective, but the cell types and mechanisms involved are not understood. To investigate whether ERβ restricted to cardiomyocytes contributes to the observed cardioprotection, we tested the effects of cardiomyocyte-specific ERβ-OE (ERβ 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 ERβ-OE and WT (wild-type) littermates were subjected to chronic anterior coronary artery ligation or sham surgery.

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.

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

Experimental studies showed that 17β-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).

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)).

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.

Amyloid-β peptides activate α1-adrenergic cardiovascular receptors.

Alzheimer disease features amyloid-β (Aβ) peptide deposition in brain and blood vessels and is associated with hypertension. Aβ peptide can cause vasoconstriction and endothelial dysfunction. We observed that Aβ peptides exert a chronotropic effect in neonatal cardiomyocytes, similar to α1-adrenergic receptor autoantibodies that we described earlier.

Ahnak1 interaction is affected by phosphorylation of Ser-296 on Cavβ₂.

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 Cavβ(2) regulatory channel subunit. Here we corroborate this functional linkage by immunocytochemistry on isolated cardiomyocytes showing co-localization of ahnak1 and Cavβ(2) in the T-tubule system.

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 β-MYH (amino acids 664-915) with alanine-mutated IQ2 domain (rβ-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.

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.

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 Cavβ(2) subunit. In this study, we addressed the question whether ahnak1/Cavβ(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 Cavβ(2) interaction as assessed by recombinant proteins.

Spinophilin is required for normal morphology, Ca(2+) homeostasis and contraction but dispensable for β-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 β-adrenergic responsiveness using a homozygous SPN knock-out mouse model (SPN-/-).

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.

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.

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 β-myosin heavy chain aa664-915 with alanine-mutated IQ2 domain (rβ-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.

A myomesin mutation associated with hypertrophic cardiomyopathy deteriorates dimerisation properties.

Myomesin plays an important structural and functional role in the M-band of striated muscles. The C-terminal domain 13 of myomesin dimerises and forms antiparallel strands which cross-link neighboring Myosin filaments and titin in the M-line of the sarcomeres. These interactions stabilise the contractile apparatus during striated muscle contraction.

Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes.

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes.

AHNAK1 and AHNAK2 are costameric proteins: AHNAK1 affects transverse skeletal muscle fiber stiffness.

The AHNAK scaffold PDZ-protein family is implicated in various cellular processes including membrane repair; however, AHNAK function and subcellular localization in skeletal muscle are unclear. We used specific AHNAK1 and AHNAK2 antibodies to analyzed the detailed localization of both proteins in mouse skeletal muscle. Co-localization of AHNAK1 and AHNAK2 with vinculin clearly demonstrates that both proteins are components of the costameric network.

Ahnak1 modulates L-type Ca(2+) channel inactivation of rodent cardiomyocytes.

Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice.

Auto-inhibitory effects of an IQ motif on protein structure and function.

The denuded IQ2 domain, i.e. myosin heavy chain not associated with regulatory light chains, exerts an inhibitory effect on myosin ATPase activity.