Understanding the molecular basis of cardiomyopathy.

Inherited cardiomyopathies are a major cause of mortality and morbidity worldwide and can be caused by mutations in a wide range of proteins located in different cellular compartments. The present review is based on Dr. Ju Chen's 2021 Robert M. Berne Distinguished Lectureship of the American Physiological Society Cardiovascular Section, in which he provided an overview of the current knowledge on the cardiomyopathy-associated proteins that have been studied in his laboratory. The review provides a general summary of the proteins in different compartments of cardiomyocytes associated with cardiomyopathies, with specific focus on the proteins that have been studied in Dr. Chen's laboratory.

Antagonizing the CX3CR1 Receptor Markedly Reduces Development of Cardiac Hypertrophy After Transverse Aortic Constriction in Mice.

ABSTRACT: Left-ventricular hypertrophy, characterized by cardiomyocyte hypertrophy, interstitial cell proliferation, and immune cell infiltration, is a high risk factor for heart failure and death. Chemokines interacting with G protein-coupled chemokine receptors probably play a role in left-ventricular hypertrophy development by promoting recruitment of activated leukocytes and modulating left-ventricular remodeling. Using the minimally invasive model of transverse aortic constriction in mice, we demonstrated that a variety of chemokine and chemokine receptor messenger Ribonucleic Acid are overexpressed in the early and late phase of hypertrophy progression. Among the chemokine receptors, Cx3cr1 and Ccr2 were most strongly overexpressed and were significantly upregulated at 3, 7, and 14 days after transverse aortic constriction. Ligands of CX3CR1 (Cx3cl1) and CCR2 (Ccl2, Ccl7, Ccl12) were significantly overexpressed in the left ventricle at the early stages after mechanical pressure overload. Pharmacological inhibition of CX3CR1 signaling using the antagonist AZD8797 led to a significant reduction of hypertrophy, whereas inhibition of CCR2 with the RS504393 antagonist did not show any effect. Furthermore, AZD8797 treatment reduced the expression of the hypertrophic marker genes Nppa and Nppb as well as the profibrotic genes Tgfb1 and Col1a1 at 14 days after transverse aortic constriction. These findings strongly suggest the involvement of the CX3CR1/CX3CL1 pathway in the pathogenesis of left-ventricular hypertrophy.

The Role of Palladin in Podocytes.

Background Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex three-dimensional morphology of podocytes depends on the actin cytoskeleton, we studied the role in podocytes of the actin bundling protein palladin, which is highly expressed therein.Methods We knocked down palladin in cultured podocytes by siRNA transfection or in zebrafish embryos by morpholino injection and studied the effects by immunofluorescence and live imaging. We also investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld-/- mice) by immunofluorescence and ultrastructural analysis and kidney biopsy specimens from patients by immunostaining for palladin.Results Compared with control-treated podocytes, palladin-knockdown podocytes had reduced actin filament staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin and α-actinin-4. Furthermore, palladin-knockdown podocytes were more susceptible to disruption of the actin cytoskeleton with cytochalasin D, latrunculin A, or jasplakinolide and showed altered migration dynamics. In zebrafish embryos, palladin knockdown compromised the morphology and dynamics of epithelial cells at an early developmental stage. Compared with PodoPalld+/+ controls, PodoPalld-/- mice developed glomeruli with a disturbed morphology, an enlarged subpodocyte space, mild effacement, and significantly reduced expression of nephrin and vinculin. Furthermore, nephrotoxic serum injection led to significantly higher levels of proteinuria in PodoPalld-/- mice than in controls. Kidney biopsy specimens from patients with diabetic nephropathy and FSGS showed downregulation of palladin in podocytes as well.Conclusions Palladin has an important role in podocyte function in vitro and in vivo.

Animal Models of Congenital Cardiomyopathies Associated With Mutations in Z-Line Proteins.

The cardiac Z-line at the boundary between sarcomeres is a multiprotein complex connecting the contractile apparatus with the cytoskeleton and the extracellular matrix. The Z-line is important for efficient force generation and transmission as well as the maintenance of structural stability and integrity. Furthermore, it is a nodal point for intracellular signaling, in particular mechanosensing and mechanotransduction. Mutations in various genes encoding Z-line proteins have been associated with different cardiomyopathies, including dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, restrictive cardiomyopathy, and left ventricular noncompaction, and mutations even within the same gene can cause widely different pathologies. Animal models have contributed to a great advancement in the understanding of the physiological function of Z-line proteins and the pathways leading from mutations in Z-line proteins to cardiomyopathy, although genotype-phenotype prediction remains a great challenge. This review presents an overview of the currently available animal models for Z-line and Z-line associated proteins involved in human cardiomyopathies with special emphasis on knock-in and transgenic mouse models recapitulating the clinical phenotypes of human cardiomyopathy patients carrying mutations in Z-line proteins. Pros and cons of mouse models will be discussed and a future outlook will be given. J. Cell. Physiol. 232: 38-52, 2017. © 2016 Wiley Periodicals, Inc.

Peptidomimetic Targeting of Cavß2 Overcomes Dysregulation of the L-Type Calcium Channel Density and Recovers Cardiac Function.

BACKGROUND: L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. METHODS: Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cavß2 chaperone regulates channel density at the plasma membrane. RESULTS: On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cavα1.2 and the Akt-dependent phosphorylation status of Cavß2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cavß2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cavα1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cavα1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cavß2, thus facilitating the chaperoning of Cavα1.2; and promotion of Cavα1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cavß2 to the nucleus, where it limits the transcription of Cavα1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cavß2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. CONCLUSIONS: We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cavß2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.

MicroRNA-133 modulates the ß1-adrenergic receptor transduction cascade.

RATIONALE: The sympathetic nervous system plays a fundamental role in the regulation of myocardial function. During chronic pressure overload, overactivation of the sympathetic nervous system induces the release of catecholamines, which activate ß-adrenergic receptors in cardiomyocytes and lead to increased heart rate and cardiac contractility. However, chronic stimulation of ß-adrenergic receptors leads to impaired cardiac function, and ß-blockers are widely used as therapeutic agents for the treatment of cardiac disease. MicroRNA-133 (miR-133) is highly expressed in the myocardium and is involved in controlling cardiac function through regulation of messenger RNA translation/stability. OBJECTIVE: To determine whether miR-133 affects ß-adrenergic receptor signaling during progression to heart failure. METHODS AND RESULTS: Based on bioinformatic analysis, ß1-adrenergic receptor (ß1AR) and other components of the ß1AR signal transduction cascade, including adenylate cyclase VI and the catalytic subunit of the cAMP-dependent protein kinase A, were predicted as direct targets of miR-133 and subsequently validated by experimental studies. Consistently, cAMP accumulation and activation of downstream targets were repressed by miR-133 overexpression in both neonatal and adult cardiomyocytes following selective ß1AR stimulation. Furthermore, gain-of-function and loss-of-function studies of miR-133 revealed its role in counteracting the deleterious apoptotic effects caused by chronic ß1AR stimulation. This was confirmed in vivo using a novel cardiac-specific TetON-miR-133 inducible transgenic mouse model. When subjected to transaortic constriction, TetON-miR-133 inducible transgenic mice maintained cardiac performance and showed attenuated apoptosis and reduced fibrosis compared with control mice. CONCLUSIONS: miR-133 controls multiple components of the ß1AR transduction cascade and is cardioprotective during heart failure.

The nebulin SH3 domain is dispensable for normal skeletal muscle structure but is required for effective active load bearing in mouse.

Nemaline myopathy (NM) is a congenital myopathy with an estimated incidence of 150,000 live births. It is caused by mutations in thin filament components, including nebulin, which accounts for about 50% of the cases. The identification of NM cases with nonsense mutations resulting in loss of the extreme C-terminal SH3 domain of nebulin suggests an important role of the nebulin SH3 domain, which is further supported by the recent demonstration of its role in IGF-1-induced sarcomeric actin filament formation through targeting of N-WASP to the Z-line. To provide further insights into the functional significance of the nebulin SH3 domain in the Z-disk and to understand the mechanisms by which truncations of nebulin lead to NM, we took two approaches: (1) an affinity-based proteomic screening to identify novel interaction partners of the nebulin SH3 domain; and (2) generation and characterization of a novel knockin mouse model with a premature stop codon in the nebulin gene, eliminating its C-terminal SH3 domain (NebΔSH3 mouse). Surprisingly, detailed analyses of NebΔSH3 mice revealed no structural or histological skeletal muscle abnormalities and no changes in gene expression or localization of interaction partners of the nebulin SH3 domain, including myopalladin, palladin, zyxin and N-WASP. Also, no significant effect on peak isometric stress production, passive tensile stress or Young's modulus was found. However, NebΔSH3 muscle displayed a slightly altered force-frequency relationship and was significantly more susceptible to eccentric contraction-induced injury, suggesting that the nebulin SH3 domain protects against eccentric contraction-induced injury and possibly plays a role in fine-tuning the excitation-contraction coupling mechanism.

Roles of Nebulin Family Members in the Heart.

The members of the nebulin protein family, including nebulin, nebulette, LASP-1, LASP-2, and N-RAP, contain various numbers of nebulin repeats and bind to actin, but are otherwise heterogeneous with regard to size, expression pattern, and function. This review focuses on the roles of nebulin family members in the heart. Nebulin is the largest member predominantly expressed in skeletal muscle, where it stretches along the thin filament. In heart, nebulin is detectable only at low levels and its absence has no apparent effects. Nebulette is similar in structure to the nebulin C-terminal Z-line region and specifically expressed in heart. Nebulette gene mutations have been identified in dilated cardiomyopathy patients and transgenic mice overexpressing nebulette mutants partially recapitulate the human pathology. In contrast, nebulette knockout mice show no functional phenotype, but exhibit Z-line widening. LASP-2 is an isoform of nebulette expressed in multiple tissues, including the heart. It is present in the Z-line and intercalated disc and able to bind and cross-link filamentous actin. LASP-1 is similar in structure to LASP-2, but expressed only in non-muscle tissue. N-RAP is present in myofibril precursors during myofibrillogenesis and thought to be involved in myofibril assembly, while it is localized at the intercalated disc in adult heart. Additional in vivo models are required to provide further insights into the functions of nebulin family members in the heart.

Neurexin-Neuroligin Synaptic Complex Regulates Schizophrenia-Related DISC1/Kal-7/Rac1 "Signalosome".

Neurexins (NXs) and neuroligins (NLs) are cell adhesion molecules that are localized at opposite sites of synaptic membranes. They interact with each other to promote the assembly, maintenance, and function of synapses in the central nervous system. Both NX and NL are cleaved from a membrane-attached intracellular domain in an activity-dependent manner, generating the soluble ectodomain of NX or NL. Expression of the NX1 and NX3 genes in the brain appears to be regulated by a schizophrenia-related protein, DISC1. Here, we show that soluble ecto-NX1ß can regulate the expression of DISC1 and induce signaling downstream of DISC1. We also show that NL1 binds to a well-characterized DISC1 interaction partner, Kal-7, and this interaction can be compromised by DISC1. Our results indicate that the NX/NL synaptic complex is intrinsically involved in the regulation of DISC1 function, thus contributing to a better understanding of the pathology of schizophrenia.

MicroRNA-133 controls cardiac hypertrophy.

Growing evidence indicates that microRNAs (miRNAs or miRs) are involved in basic cell functions and oncogenesis. Here we report that miR-133 has a critical role in determining cardiomyocyte hypertrophy. We observed decreased expression of both miR-133 and miR-1, which belong to the same transcriptional unit, in mouse and human models of cardiac hypertrophy. In vitro overexpression of miR-133 or miR-1 inhibited cardiac hypertrophy. In contrast, suppression of miR-133 by 'decoy' sequences induced hypertrophy, which was more pronounced than that after stimulation with conventional inducers of hypertrophy. In vivo inhibition of miR-133 by a single infusion of an antagomir caused marked and sustained cardiac hypertrophy. We identified specific targets of miR-133: RhoA, a GDP-GTP exchange protein regulating cardiac hypertrophy; Cdc42, a signal transduction kinase implicated in hypertrophy; and Nelf-A/WHSC2, a nuclear factor involved in cardiogenesis. Our data show that miR-133, and possibly miR-1, are key regulators of cardiac hypertrophy, suggesting their therapeutic application in heart disease.

Lung-to-Heart Nano-in-Micro Peptide Promotes Cardiac Recovery in a Pig Model of Chronic Heart Failure.

BACKGROUND: The lack of disease-modifying drugs is one of the major unmet needs in patients with heart failure (HF). Peptides are highly selective molecules with the potential to act directly on cardiomyocytes. However, a strategy for effective delivery of therapeutics to the heart is lacking. OBJECTIVES: In this study, the authors sought to assess tolerability and efficacy of an inhalable lung-to-heart nano-in-micro technology (LungToHeartNIM) for cardiac-specific targeting of a mimetic peptide (MP), a first-in-class for modulating impaired L-type calcium channel (LTCC) trafficking, in a clinically relevant porcine model of HF. METHODS: Heart failure with reduced ejection fraction (HFrEF) was induced in Göttingen minipigs by means of tachypacing over 6 weeks. In a setting of overt HFrEF (left ventricular ejection fraction [LVEF] 30% ± 8%), animals were randomized and treatment was started after 4 weeks of tachypacing. HFrEF animals inhaled either a dry powder composed of mannitol-based microparticles embedding biocompatible MP-loaded calcium phosphate nanoparticles (dpCaP-MP) or the LungToHeartNIM only (dpCaP without MP). Efficacy was evaluated with the use of echocardiography, invasive hemodynamics, and biomarker assessment. RESULTS: DpCaP-MP inhalation restored systolic function, as shown by an absolute LVEF increase over the treatment period of 17% ± 6%, while reversing cardiac remodeling and reducing pulmonary congestion. The effect was recapitulated ex vivo in cardiac myofibrils from treated HF animals. The treatment was well tolerated, and no adverse events occurred. CONCLUSIONS: The overall tolerability of LungToHeartNIM along with the beneficial effects of the LTCC modulator point toward a game-changing treatment for HFrEF patients, also demonstrating the effective delivery of a therapeutic peptide to the diseased heart.

A matter of balance: role of neurexin and neuroligin at the synapse.

Neurexins and neuroligins are synaptic cell adhesion molecules. Neurexins are primary located on the presynaptic membrane, whereas neuroligins are strictly postsynaptic proteins. Since their discovery, the knowledge of neurexins and neuroligins has expanded, implicating them in various neuronal processes, including the differentiation, maturation, stabilization, and plasticity of both inhibitory and excitatory synapses. Here, we review the most recent results regarding the structure and function of these cell adhesion molecules.

Whole Genome Expression Profiling of Semitendinosus Tendons from Children with Diplegic and Tetraplegic Cerebral Palsy.

Cerebral palsy (CP) is the most common movement disorder in children, with a prevalence ranging from 1.5 to 4 per 1000 live births. CP is caused by a non-progressive lesion of the developing brain, leading to progressive alterations of the musculoskeletal system, including spasticity, often leading to the development of fixed contractures, necessitating tendon lengthening surgery. Total RNA-sequencing analysis was performed on semitendinosus tendons from diplegic and tetraplegic CP patients subjected to tendon lengthening surgery compared to control patients undergoing anterior cruciate ligament reconstructive surgery. Tetraplegic CP patients showed increased expression of genes implicated in collagen synthesis and extracellular matrix (ECM) turnover, while only minor changes were observed in diplegic CP patients. In addition, tendons from tetraplegic CP patients showed an enrichment for upregulated genes involved in vesicle-mediated transport and downregulated genes involved in cytokine and apoptotic signaling. Overall, our results indicate increased ECM turnover with increased net synthesis of collagen in tetraplegic CP patients without activation of inflammatory and apoptotic pathways, similar to observations in athletes where ECM remodeling results in increased tendon stiffness and tensile strength. Nevertheless, the resulting increased tendon stiffness is an important issue in clinical practice, where surgery is often required to restore joint mobility.

Ablation of palladin in adult heart causes dilated cardiomyopathy associated with intercalated disc abnormalities.

Palladin (PALLD) belongs to the PALLD/myopalladin (MYPN)/myotilin family of actin-associated immunoglobulin-containing proteins in the sarcomeric Z-line. PALLD is ubiquitously expressed in several isoforms, and its longest 200 kDa isoform, predominantly expressed in striated muscle, shows high structural homology to MYPN. MYPN gene mutations are associated with human cardiomyopathies, whereas the role of PALLD in the heart has remained unknown, partly due to embryonic lethality of PALLD knockout mice. In a yeast two-hybrid screening, CARP/Ankrd1 and FHOD1 were identified as novel interaction partners of PALLD's N-terminal region. To study the role of PALLD in the heart, we generated conditional (cPKO) and inducible (cPKOi) cardiomyocyte-specific PALLD knockout mice. While cPKO mice exhibited no pathological phenotype, ablation of PALLD in adult cPKOi mice caused progressive cardiac dilation and systolic dysfunction, associated with reduced cardiomyocyte contractility, intercalated disc abnormalities, and fibrosis, demonstrating that PALLD is essential for normal cardiac function. Double cPKO and MYPN knockout (MKO) mice exhibited a similar phenotype as MKO mice, suggesting that MYPN does not compensate for the loss of PALLD in cPKO mice. Altered transcript levels of MYPN and PALLD isoforms were found in myocardial tissue from human dilated and ischemic cardiomyopathy patients, whereas their protein expression levels were unaltered.

Video analysis of ex vivo beating hearts during preservation on the TransMedics® organ care system.

Background: Reliable biomarkers for assessing the viability of the donor hearts undergoing ex vivo perfusion remain elusive. A unique feature of normothermic ex vivo perfusion on the TransMedics® Organ Care System (OCS™) is that the donor heart is maintained in a beating state throughout the preservation period. We applied a video algorithm for an in vivo assessment of cardiac kinematics, video kinematic evaluation (Vi.Ki.E.), to the donor hearts undergoing ex vivo perfusion on the OCS™ to assess the feasibility of applying this algorithm in this setting. Methods: Healthy donor porcine hearts (n = 6) were procured from Yucatan pigs and underwent 2 h of normothermic ex vivo perfusion on the OCS™ device. During the preservation period, serial high-resolution videos were captured at 30 frames per second. Using Vi.Ki.E., we assessed the force, energy, contractility, and trajectory parameters of each heart. Results: There were no significant changes in any of the measured parameters of the heart on the OCS™ device over time as judged by linear regression analysis. Importantly, there were no significant changes in contractility during the duration of the preservation period (time 0-30 min, 918 ± 430 px/s; time 31-60 min, 1,386 ± 603 px/s; time 61-90 min, 1,299 ± 617 px/s; time 91-120 min, 1,535 ± 728 px/s). Similarly, there were no significant changes in the force, energy, or trajectory parameters. Post-transplantation echocardiograms demonstrated robust contractility of each allograft. Conclusion: Vi.Ki.E. assessment of the donor hearts undergoing ex vivo perfusion is feasible on the TransMedics OCS™, and we observed that the donor hearts maintain steady kinematic measurements throughout the duration.

Unexpectedly low mutation rates in beta-myosin heavy chain and cardiac myosin binding protein genes in Italian patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. Fourteen sarcomeric and sarcomere-related genes have been implicated in HCM etiology, those encoding ß-myosin heavy chain (MYH7) and cardiac myosin binding protein C (MYBPC3) reported as the most frequently mutated: in fact, these account for around 50% of all cases related to sarcomeric gene mutations, which are collectively responsible for approximately 70% of all HCM cases. Here, we used denaturing high-performance liquid chromatography followed by bidirectional sequencing to screen the coding regions of MYH7 and MYBPC3 in a cohort (n = 125) of Italian patients presenting with HCM. We found 6 MHY7 mutations in 9/125 patients and 18 MYBPC3 mutations in 19/125 patients. Of the three novel MYH7 mutations found, two were missense, and one was a silent mutation; of the eight novel MYBPC3 mutations, one was a substitution, three were stop codons, and four were missense mutations. Thus, our cohort of Italian HCM patients did not harbor the high frequency of mutations usually found in MYH7 and MYBPC3. This finding, coupled to the clinical diversity of our cohort, emphasizes the complexity of HCM and the need for more inclusive investigative approaches in order to fully understand the pathogenesis of this disease.

Cardiac-specific ablation of Cypher leads to a severe form of dilated cardiomyopathy with premature death.

Accumulating data suggest a link between alterations/deficiencies in cytoskeletal proteins and the progression of cardiomyopathy and heart failure, although the molecular basis for this link remains unclear. Cypher/ZASP is a cytoskeletal protein localized in the sarcomeric Z-line. Mutations in its encoding gene have been identified in patients with isolated non-compaction of the left ventricular myocardium, dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy. To explore the role of Cypher in myocardium and to better understand molecular mechanisms by which mutations in cypher cause cardiomyopathy, we utilized a conditional approach to knockout Cypher, specially in either developing or adult myocardium. Cardiac-specific Cypher knockout (CKO) mice developed a severe form of DCM with disrupted cardiomyocyte ultrastructure and decreased cardiac function, which eventually led to death before 23 weeks of age. A similar phenotype was observed in inducible cardiac-specific CKO mice in which Cypher was specifically ablated in adult myocardium. In both cardiac-specific CKO models, ERK and Stat3 signaling pathways were augmented. Finally, we demonstrate the specific binding of Cypher's PDZ domain to the C-terminal region of both calsarcin-1 and myotilin within the Z-line. In conclusion, our studies suggest that (i) Cypher plays a pivotal role in maintaining adult cardiac structure and cardiac function through protein-protein interactions with other Z-line proteins, (ii) myocardial ablation of Cypher results in DCM with premature death and (iii) specific signaling pathways participate in Cypher mutant-mediated dysfunction of the heart, and may in concert facilitate the progression to heart failure.

Nebulin-deficient mice exhibit shorter thin filament lengths and reduced contractile function in skeletal muscle.

Nebulin is a giant modular sarcomeric protein that has been proposed to play critical roles in myofibrillogenesis, thin filament length regulation, and muscle contraction. To investigate the functional role of nebulin in vivo, we generated nebulin-deficient mice by using a Cre knock-in strategy. Lineage studies utilizing this mouse model demonstrated that nebulin is expressed uniformly in all skeletal muscles. Nebulin-deficient mice die within 8-11 d after birth, with symptoms including decreased milk intake and muscle weakness. Although myofibrillogenesis had occurred, skeletal muscle thin filament lengths were up to 25% shorter compared with wild type, and thin filaments were uniform in length both within and between muscle types. Ultrastructural studies also demonstrated a critical role for nebulin in the maintenance of sarcomeric structure in skeletal muscle. The functional importance of nebulin in skeletal muscle function was revealed by isometric contractility assays, which demonstrated a dramatic reduction in force production in nebulin-deficient skeletal muscle.

Myopalladin knockout mice develop cardiac dilation and show a maladaptive response to mechanical pressure overload.

Myopalladin (MYPN) is a striated muscle-specific immunoglobulin domain-containing protein located in the sarcomeric Z-line and I-band. MYPN gene mutations are causative for dilated (DCM), hypertrophic, and restrictive cardiomyopathy. In a yeast two-hybrid screening, MYPN was found to bind to titin in the Z-line, which was confirmed by microscale thermophoresis. Cardiac analyses of MYPN knockout (MKO) mice showed the development of mild cardiac dilation and systolic dysfunction, associated with decreased myofibrillar isometric tension generation and increased resting tension at longer sarcomere lengths. MKO mice exhibited a normal hypertrophic response to transaortic constriction (TAC), but rapidly developed severe cardiac dilation and systolic dysfunction, associated with fibrosis, increased fetal gene expression, higher intercalated disc fold amplitude, decreased calsequestrin-2 protein levels, and increased desmoplakin and SORBS2 protein levels. Cardiomyocyte analyses showed delayed Ca2+ release and reuptake in unstressed MKO mice as well as reduced Ca2+ spark amplitude post-TAC, suggesting that altered Ca2+ handling may contribute to the development of DCM in MKO mice.

Nebulin plays a direct role in promoting strong actin-myosin interactions.

The role of the actin filament-associated protein nebulin on mechanical and kinetic properties of the actomyosin motor was investigated in skeletal muscle of wild-type (wt) and nebulin-deficient (nebulin(-)(/)(-)) mice that were 1 d old, an age at which sarcomeric structure is still well preserved. In Ca2+-activated skinned fibers from psoas muscle, we determined the Ca2+ dependence of isometric force and stiffness, the rate of force redevelopment after unloaded shortening (k(TR)), the power during isotonic shortening, and the unloaded shortening velocity (V(0)). Our results show a 65% reduction in isometric force in nebulin(-)(/)(-) fibers at saturating [Ca2+], whereas neither thin-filament length nor the Ca2+ sensitivity of the contractile system is affected. Stiffness measurements indicate that the reduction in isometric force is due to a reduction in the number of actin-attached myosin motors, whereas the force of the motor is unchanged. Furthermore, in nebulin(-)(/)(-) fibers, k(TR) is decreased by 57%, V(0) is increased by 63%, and the maximum power is decreased by 80%. These results indicate that, in the absence of nebulin, the attachment probability of the myosin motors to actin is decreased, revealing a direct role for nebulin in promoting strong actomyosin interactions responsible for force and power production.