Differential sex-dependent susceptibility to diastolic dysfunction and arrhythmia in cardiomyocytes from obese diabetic HFpEF model.

AIM: Sex-differences in heart failure with preserved ejection fraction (HFpEF) are important, but key mechanisms involved are incompletely understood. While animal models can inform about sex-dependent cellular and molecular changes, many previous preclinical HFpEF models have failed to recapitulate sex-dependent characteristics of human HFpEF. We tested for sex-differences in HFpEF using a two-hit mouse model (leptin receptor-deficient db/db mice plus aldosterone infusion for 4 weeks; db/db+Aldo). METHODS AND RESULTS: We performed echocardiography, electrophysiology, intracellular Ca2+ imaging, and protein analysis. Female HFpEF mice exhibited more severe diastolic dysfunction in line with increased titin N2B isoform expression and PEVK element phosphorylation, and reduced troponin-I phosphorylation. Female HFpEF mice had lower BNP levels than males despite similar comorbidity burden (obesity, diabetes) and cardiac hypertrophy in both sexes. Male HFpEF mice were more susceptible to cardiac alternans. Male HFpEF cardiomyocytes (versus female) exhibited higher diastolic [Ca2+], slower Ca2+ transient decay, reduced L-type Ca2+ current, more pronounced enhancement of the late Na+ current, and increased short-term variability of action potential duration (APD). However, male and female HFpEF myocytes showed similar downregulation of inward rectifier and transient outward K+ currents, APD prolongation, and frequency of delayed afterdepolarizations. Inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed all pathological APD changes in HFpEF in both sexes, and empagliflozin pretreatment mimicked these effects of CaMKII inhibition. Vericiguat had only slight benefits, and these effects were larger in HFpEF females. CONCLUSION: We conclude that the db/db+Aldo preclinical HFpEF murine model recapitulates key sex-specific mechanisms in HFpEF and provides mechanistic insights into impaired excitation-contraction coupling and sex-dependent differential arrhythmia susceptibility in HFpEF with potential therapeutic implications. In male HFpEF myocytes, altered Ca2+ handling and electrophysiology aligned with diastolic dysfunction and arrhythmias, while worse diastolic dysfunction in females may depend more on altered myofilaments properties.

Characterization of NEB pathogenic variants in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects.

Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

Characterization of NEB mutations in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects.

Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Mutations in the nebulin gene ( NEB ) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking therapies targeting the underlying pathological mechanisms. In this study, we examined a cohort of ten NEM2 patients, each with unique mutations, aiming to understand their impact on mRNA, protein, and functional levels. Results show that truncation mutations affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with splicing mutations which is expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with relatively normal nebulin levels and others with markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, and a positive relation between the reduction in nebulin level and the reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a duplication mutation in nebulin that resulted in a larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type I muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87-318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from splicing mutations. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

Proteomic and phosphoproteomic profiling in heart failure with preserved ejection fraction (HFpEF).

Although the prevalence of heart failure with preserved ejection fraction (HFpEF) is increasing, evidence-based therapies for HFpEF remain limited, likely due to an incomplete understanding of this disease. This study sought to identify the cardiac-specific features of protein and phosphoprotein changes in a murine model of HFpEF using mass spectrometry. HFpEF mice demonstrated moderate hypertension, left ventricle (LV) hypertrophy, lung congestion and diastolic dysfunction. Proteomics analysis of the LV tissue showed that 897 proteins were differentially expressed between HFpEF and Sham mice. We observed abundant changes in sarcomeric proteins, mitochondrial-related proteins, and NAD-dependent protein deacetylase sirtuin-3 (SIRT3). Upregulated pathways by GSEA analysis were related to immune modulation and muscle contraction, while downregulated pathways were predominantly related to mitochondrial metabolism. Western blot analysis validated SIRT3 downregulated cardiac expression in HFpEF vs. Sham (0.8 ± 0.0 vs. 1.0 ± 0.0; P < 0.001). Phosphoproteomics analysis showed that 72 phosphosites were differentially regulated between HFpEF and Sham LV. Aberrant phosphorylation patterns mostly occurred in sarcomere proteins and nuclear-localized proteins associated with contractile dysfunction and cardiac hypertrophy. Seven aberrant phosphosites were observed at the z-disk binding region of titin. Additional agarose gel analysis showed that while total titin cardiac expression remained unaltered, its stiffer N2B isoform was significantly increased in HFpEF vs. Sham (0.144 ± 0.01 vs. 0.127 ± 0.01; P < 0.05). In summary, this study demonstrates marked changes in proteins related to mitochondrial metabolism and the cardiac contractile apparatus in HFpEF. We propose that SIRT3 may play a role in perpetuating these changes and may be a target for drug development in HFpEF.

Removal of MuRF1 Increases Muscle Mass in Nemaline Myopathy Models, but Does Not Provide Functional Benefits.

Nemaline myopathy (NM) is characterized by skeletal muscle weakness and atrophy. No curative treatments exist for this debilitating disease. NM is caused by mutations in proteins involved in thin-filament function, turnover, and maintenance. Mutations in nebulin, encoded by NEB, are the most common cause. Skeletal muscle atrophy is tightly linked to upregulation of MuRF1, an E3 ligase, that targets proteins for proteasome degradation. Here, we report a large increase in MuRF1 protein levels in both patients with nebulin-based NM, also named NEM2, and in mouse models of the disease. We hypothesized that knocking out MuRF1 in animal models of NM with muscle atrophy would ameliorate the muscle deficits. To test this, we crossed MuRF1 KO mice with two NEM2 mouse models, one with the typical form and the other with the severe form. The crosses were viable, and muscles were studied in mice at 3 months of life. Ultrastructural examination of gastrocnemius muscle lacking MuRF1 and with severe NM revealed a small increase in vacuoles, but no significant change in the myofibrillar fractional area. MuRF1 deficiency led to increased weights of various muscle types in the NM models. However, this increase in muscle size was not associated with increased in vivo or in vitro force production. We conclude that knocking out MuRF1 in NEM2 mice increases muscle size, but does not improve muscle function.

HDAC6 modulates myofibril stiffness and diastolic function of the heart.

Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases.

Nebulin and Lmod2 are critical for specifying thin-filament length in skeletal muscle.

Regulating the thin-filament length in muscle is crucial for controlling the number of myosin motors that generate power. The giant protein nebulin forms a long slender filament that associates along the length of the thin filament in skeletal muscle with functions that remain largely obscure. Here nebulin's role in thin-filament length regulation was investigated by targeting entire super-repeats in the Neb gene; nebulin was either shortened or lengthened by 115 nm. Its effect on thin-filament length was studied using high-resolution structural and functional techniques. Results revealed that thin-filament length is strictly regulated by the length of nebulin in fast muscles. Nebulin's control is less tight in slow muscle types where a distal nebulin-free thin-filament segment exists, the length of which was found to be regulated by leiomodin-2 (Lmod2). We propose that strict length control by nebulin promotes high-speed shortening and that dual-regulation by nebulin/Lmod2 enhances contraction efficiency.

Triggering typical nemaline myopathy with compound heterozygous nebulin mutations reveals myofilament structural changes as pathomechanism.

Nebulin is a giant protein that winds around the actin filaments in the skeletal muscle sarcomere. Compound-heterozygous mutations in the nebulin gene (NEB) cause typical nemaline myopathy (NM), a muscle disorder characterized by muscle weakness with limited treatment options. We created a mouse model with a missense mutation p.Ser6366Ile and a deletion of NEB exon 55, the Compound-Het model that resembles typical NM. We show that Compound-Het mice are growth-retarded and have muscle weakness. Muscles have a reduced myofibrillar fractional-area and sarcomeres are disorganized, contain rod bodies, and have longer thin filaments. In contrast to nebulin-based severe NM where haplo-insufficiency is the disease driver, Compound-Het mice express normal amounts of nebulin. X-ray diffraction revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased. The unique disease mechanism of nebulin-based typical NM reveals novel therapeutic targets.

Phosphodiesterase 9a Inhibition in Mouse Models of Diastolic Dysfunction.

BACKGROUND: Low myocardial cGMP-PKG (cyclic guanosine monophosphate-protein kinase G) activity has been associated with increased cardiomyocyte diastolic stiffness in heart failure with preserved ejection fraction. Cyclic guanosine monophosphate is mainly hydrolyzed by PDE (phosphodiesterases) 5a and 9a. Importantly, PDE9a expression has been reported to be upregulated in human heart failure with preserved ejection fraction myocardium and chronic administration of a PDE9a inhibitor reverses preestablished cardiac hypertrophy and systolic dysfunction in mice subjected to transverse aortic constriction (TAC). We hypothesized that inhibiting PDE9a activity ameliorates diastolic dysfunction. METHODS: To examine the effect of chronic PDE9a inhibition, 2 diastolic dysfunction mouse models were studied: (1) TAC-deoxycorticosterone acetate and (2) Leprdb/db. PDE9a inhibitor (5 and 8 mg/kg per day) was administered to the mice via subcutaneously implanted osmotic minipumps for 28 days. The effect of acute PDE9a inhibition was investigated in intact cardiomyocytes isolated from TAC-deoxycorticosterone acetate mice. Atrial natriuretic peptide together with PDE9a inhibitor were administered to the isolated intact cardiomyocytes through the cell perfusate. RESULTS: For acute inhibition, no cellular stiffness reduction was found, whereas chronic PDE9a inhibition resulted in reduced left ventricular chamber stiffness in TAC-deoxycorticosterone acetate, but not in Leprdb/db mice. Passive cardiomyocyte stiffness was reduced by chronic PDE9a inhibition, with no differences in myocardial fibrosis or cardiac morphometry. PDE9a inhibition increased the ventricular-arterial coupling ratio, reflecting impaired systolic function. CONCLUSIONS: Chronic PDE9a inhibition lowers left ventricular chamber stiffness in TAC-deoxycorticosterone acetate mice. However, the usefulness of PDE9a inhibition to treat high-diastolic stiffness may be limited as the required PDE9a inhibitor dose also impairs systolic function, observed as a decline in ventricular-arterial coordination, in this model.

Correction to: Expressing a Z-disk nebulin fragment innebulin-deficient mouse muscle: effects on muscle structure and function.

Following the publication of this paper [1], it was brought to the authors' attention that one of the contributing authors was left off of the paper. The authors apologize for the unfortunate oversight. In this correction paper, they have included Dr. Paola Tonino in the author list section.

Expressing a Z-disk nebulin fragment in nebulin-deficient mouse muscle: effects on muscle structure and function.

BACKGROUND: Nebulin is a critical thin filament-binding protein that spans from the Z-disk of the skeletal muscle sarcomere to near the pointed end of the thin filament. Its massive size and actin-binding property allows it to provide the thin filaments with structural and regulatory support. When this protein is lost, nemaline myopathy occurs. Nemaline myopathy causes severe muscle weakness as well as structural defects on a sarcomeric level. There is no known cure for this disease. METHODS: We studied whether sarcomeric structure and function can be improved by introducing nebulin's Z-disk region into a nebulin-deficient mouse model (Neb cKO) through adeno-associated viral (AAV) vector therapy. Following this treatment, the structural and functional characteristics of both vehicle-treated and AAV-treated Neb cKO and control muscles were studied. RESULTS: Intramuscular injection of this AAV construct resulted in a successful expression of the Z-disk fragment within the target muscles. This expression was significantly higher in Neb cKO mice than control mice. Analysis of protein expression revealed that the nebulin fragment was localized exclusively to the Z-disks and that Neb cKO expressed the nebulin fragment at levels comparable to the level of full-length nebulin in control mice. Additionally, the Z-disk fragment displaced full-length nebulin in control mice, resulting in nemaline rod body formation and a worsening of muscle function. Neb cKO mice experienced a slight functional benefit from the AAV treatment, with a small increase in force and fatigue resistance. Disease progression was also slowed as indicated by improved muscle structure and myosin isoform expression. CONCLUSIONS: This study reveals that nebulin fragments are well-received by nebulin-deficient mouse muscles and that limited functional benefits are achievable.

In vivo characterization of skeletal muscle function in nebulin-deficient mice.

INTRODUCTION: The conditional nebulin knockout mouse is a new model mimicking nemaline myopathy, a rare disease characterized by muscle weakness and rods within muscle fibers. We investigated the impact of nebulin (NEB) deficiency on muscle function in vivo. METHODS: Conditional nebulin knockout mice and control littermates were studied at 10 to 12 months. Muscle function (force and fatigue) and anatomy (muscles volume and fat content) were measured in vivo. Myosin heavy chain (MHC) composition and nebulin (NEB) protein expression were assessed by protein electrophoresis. RESULTS: Conditional nebulin knockout mice displayed a lower NEB level (-90%) leading to a 40% and 45% reduction in specific maximal force production and muscles volume, respectively. Nebulin deficiency was also associated with higher resistance to fatigue and increased MHC I content. DISCUSSION: Adult nebulin-deficient mice displayed severe muscle atrophy and weakness in vivo related to a low NEB content but an improved fatigue resistance due to a slower contractile phenotype.