Metabolic Flexibility of the Heart: The Role of Fatty Acid Metabolism in Health, Heart Failure, and Cardiometabolic Diseases.

This comprehensive review explores the critical role of fatty acid (FA) metabolism in cardiac diseases, particularly heart failure (HF), and the implications for therapeutic strategies. The heart's reliance on ATP, primarily sourced from mitochondrial oxidative metabolism, underscores the significance of metabolic flexibility, with fatty acid oxidation (FAO) being a dominant source. In HF, metabolic shifts occur with an altered FA uptake and FAO, impacting mitochondrial function and contributing to disease progression. Conditions like obesity and diabetes also lead to metabolic disturbances, resulting in cardiomyopathy marked by an over-reliance on FAO, mitochondrial dysfunction, and lipotoxicity. Therapeutic approaches targeting FA metabolism in cardiac diseases have evolved, focusing on inhibiting or stimulating FAO to optimize cardiac energetics. Strategies include using CPT1A inhibitors, using PPARα agonists, and enhancing mitochondrial biogenesis and function. However, the effectiveness varies, reflecting the complexity of metabolic remodeling in HF. Hence, treatment strategies should be individualized, considering that cardiac energy metabolism is intricate and tightly regulated. The therapeutic aim is to optimize overall metabolic function, recognizing the pivotal role of FAs and the need for further research to develop effective therapies, with promising new approaches targeting mitochondrial oxidative metabolism and FAO that improve cardiac function.

Students at a crossroad: A cross-sectional survey gauging the impact of COVID-19 on medical and biomedical graduates in the United States and Sweden.

Graduate programs in medicine and biomedical sciences have been severely impacted by the SARS-CoV-2/COVID-19 pandemic over the last 2 years. Following 2 years since beginning of the pandemic, data on student support, educational and academic performance as well as sentiment on changes to educational programs are starting to emerge. We performed and compared results of two cross-sectional surveys of Swedish and U.S.-based medical and biomedical graduate students on how the pandemic has affected their studies, research productivity and career trajectory. Students were also asked to assess support provided by the university and supervisors. The surveys also captured student demographics and a range of other factors, such as pressures brought on by caretaking and financial responsibilities. We analyzed answers from 264 and 106 students attending graduate programs in universities in Sweden and the United States, respectively. U.S.-based students faced more severe restrictions on their research program compared to students in Sweden, reporting more delays in productivity, scientific output and graduation, and increased worries about their career trajectory. Swedish students had more caretaking responsibilities, although these did not cause any delays in graduation. While support by universities and supervisors was comparable between the countries, financial worries and mental health concerns were particularly prominent in the U.S. cohort. Student performance and outlook was hugely dependent on the breadth of the restrictions and the available support. Besides the governmental and university-led approach to counter the pandemic, societal differences also played a role in how well students were handling effects of the pandemic.

Lipoxins reduce obesity-induced adipose tissue inflammation in 3D-cultured human adipocytes and explant cultures.

Adipose tissue inflammation drives obesity-related cardiometabolic diseases. Enhancing endogenous resolution mechanisms through administration of lipoxin A4, a specialized pro-resolving lipid mediator, was shown to reduce adipose inflammation and subsequently protects against obesity-induced systemic disease in mice. Here, we demonstrate that lipoxins reduce inflammation in 3D-cultured human adipocytes and adipose tissue explants from obese patients. Approximately 50% of patients responded particularly well to lipoxins by reducing inflammatory cytokines and promoting an anti-inflammatory M2 macrophage phenotype. Responding patients were characterized by elevated systemic levels of C-reactive protein, which causes inflammation in cultured human adipocytes. Responders appeared more prone to producing anti-inflammatory oxylipins and displayed elevated prostaglandin D2 levels, which has been interlinked with transcription of lipoxin-generating enzymes. Using explant cultures, this study provides the first proof-of-concept evidence supporting the therapeutic potential of lipoxins in reducing human adipose tissue inflammation. Our data further indicate that lipoxin treatment may require a tailored personalized-medicine approach.

Impaired Intracellular Ca2+ Dynamics, M-Band and Sarcomere Fragility in Skeletal Muscles of Obscurin KO Mice.

Obscurin is a giant sarcomeric protein expressed in striated muscles known to establish several interactions with other proteins of the sarcomere, but also with proteins of the sarcoplasmic reticulum and costameres. Here, we report experiments aiming to better understand the contribution of obscurin to skeletal muscle fibers, starting with a detailed characterization of the diaphragm muscle function, which we previously reported to be the most affected muscle in obscurin (Obscn) KO mice. Twitch and tetanus tension were not significantly different in the diaphragm of WT and Obscn KO mice, while the time to peak (TTP) and half relaxation time (HRT) were prolonged. Differences in force-frequency and force-velocity relationships and an enhanced fatigability are observed in an Obscn KO diaphragm with respect to WT controls. Voltage clamp experiments show that a sarcoplasmic reticulum's Ca2+ release and SERCA reuptake rates were decreased in muscle fibers from Obscn KO mice, suggesting that an impairment in intracellular Ca2+ dynamics could explain the observed differences in the TTP and HRT in the diaphragm. In partial contrast with previous observations, Obscn KO mice show a normal exercise tolerance, but fiber damage, the altered sarcomere ultrastructure and M-band disarray are still observed after intense exercise.

The titin N2B and N2A regions: biomechanical and metabolic signaling hubs in cross-striated muscles.

Muscle specific signaling has been shown to originate from myofilaments and their associated cellular structures, including the sarcomeres, costameres or the cardiac intercalated disc. Two signaling hubs that play important biomechanical roles for cardiac and/or skeletal muscle physiology are the N2B and N2A regions in the giant protein titin. Prominent proteins associated with these regions in titin are chaperones Hsp90 and αB-crystallin, members of the four-and-a-half LIM (FHL) and muscle ankyrin repeat protein (Ankrd) families, as well as thin filament-associated proteins, such as myopalladin. This review highlights biological roles and properties of the titin N2B and N2A regions in health and disease. Special emphasis is placed on functions of Ankrd and FHL proteins as mechanosensors that modulate muscle-specific signaling and muscle growth. This region of the sarcomere also emerged as a hotspot for the modulation of passive muscle mechanics through altered titin phosphorylation and splicing, as well as tethering mechanisms that link titin to the thin filament system.

Challenges in PhD education due to COVID-19 - disrupted supervision or business as usual: a cross-sectional survey of Swedish biomedical sciences graduate students.

BACKGROUND: It remains unclear to what extent the SARS-CoV-2/COVID-19 pandemic disrupted the normal progression of biomedical and medical science graduate programs and if there was a lasting impact on the quality and quantity of supervision of PhD-students. To date, multiple editorials and commentaries indicate the severity of the disruption without providing sufficient evidence with quantifiable data. METHODS: An online survey was submitted to the administrative offices of biomedical and medical PhD-programs at eight major universities in Sweden to gauge the impact of the pandemic on the students. It consisted of multiple-choice and open-ended questions where students could provide examples of positive and/or negative supervision strategies. Open answered questions were coded as either examples of positive or negative support. RESULTS: PhD students were divided into two groups: those with improved or unchanged supervision during the pandemic (group 1, n = 185), versus those whose supervision worsened (group 2, n = 69). Group 1 received more help from supervisors and more frequent supervision via both online and alternative platforms (email/messages and telephone). There was no significant difference in educational-stage, gender or caretaking responsibilities between the groups. CONCLUSIONS: It is important for the scientific community to learn how to provide the best possible supervision for PhD students during the pandemic. Our data suggests that more frequent supervision, and using a diverse array of meeting platforms is helpful. In addition, it is important for the students to feel that they have their supervisor's emotional support. Several students also expressed that they would benefit from an extension of their PhD programs due to delays caused by the pandemic.

Desmosomal COP9 regulates proteome degradation in arrhythmogenic right ventricular dysplasia/cardiomyopathy.

Dysregulated protein degradative pathways are increasingly recognized as mediators of human disease. This mechanism may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication, as destabilization/breakdown of the desmosomal proteome is a hallmark of genetic-based desmosomal-targeted diseases, such as the cardiac disease arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here, we uncovered a cardiac constitutive photomorphogenesis 9 (COP9) desmosomal resident protein complex, composed of subunit 6 of the COP9 signalosome (CSN6), that enzymatically restricted neddylation and targeted desmosomal proteome degradation. CSN6 binding, localization, levels, and function were affected in hearts of classic mouse and human models of ARVD/C affected by desmosomal loss and mutations, respectively. Loss of desmosomal proteome degradation control due to junctional reduction/loss of CSN6 and human desmosomal mutations destabilizing junctional CSN6 were also sufficient to trigger ARVD/C in mice. We identified a desmosomal resident regulatory complex that restricted desmosomal proteome degradation and disease.

The N2A region of titin has a unique structural configuration.

The N2A segment of titin is a main signaling hub in the sarcomeric I-band that recruits various signaling factors and processing enzymes. It has also been proposed to play a role in force production through its Ca2+-regulated association with actin. However, the molecular basis by which N2A performs these functions selectively within the repetitive and extensive titin chain remains poorly understood. Here, we analyze the structure of N2A components and their association with F-actin. Specifically, we characterized the structure of its Ig domains by elucidating the atomic structure of the I81-I83 tandem using x-ray crystallography and computing a homology model for I80. Structural data revealed these domains to present heterogeneous and divergent Ig folds, where I81 and I83 have unique loop structures. Notably, the I81-I83 tandem has a distinct rotational chain arrangement that confers it a unique multi-domain topography. However, we could not identify specific Ca2+-binding sites in these Ig domains, nor evidence of the association of titin N2A components with F-actin in transfected C2C12 myoblasts or C2C12-derived myotubes. In addition, F-actin cosedimentation assays failed to reveal binding to N2A. We conclude that N2A has a unique architecture that predictably supports its selective recruitment of binding partners in signaling, but that its mechanical role through interaction with F-actin awaits validation.

Exploring Obscurin and SPEG Kinase Biology.

Three members of the obscurin protein family that contain tandem kinase domains with important signaling functions for cardiac and striated muscles are the giant protein obscurin, its obscurin-associated kinase splice isoform, and the striated muscle enriched protein kinase (SPEG). While there is increasing evidence for the specific roles that each individual kinase domain plays in cross-striated muscles, their biology and regulation remains enigmatic. Our present study focuses on kinase domain 1 and the adjacent low sequence complexity inter-kinase domain linker in obscurin and SPEG. Using Phos-tag gels, we show that the linker in obscurin contains several phosphorylation sites, while the same region in SPEG remained unphosphorylated. Our homology modeling, mutational analysis and molecular docking demonstrate that kinase 1 in obscurin harbors all key amino acids important for its catalytic function and that actions of this domain result in autophosphorylation of the protein. Our bioinformatics analyses also assign a list of putative substrates for kinase domain 1 in obscurin and SPEG, based on the known and our newly proposed phosphorylation sites in muscle proteins, including obscurin itself.

Molecular Characterisation of Titin N2A and Its Binding of CARP Reveals a Titin/Actin Cross-linking Mechanism.

Striated muscle responds to mechanical overload by rapidly up-regulating the expression of the cardiac ankyrin repeat protein, CARP, which then targets the sarcomere by binding to titin N2A in the I-band region. To date, the role of this interaction in the stress response of muscle remains poorly understood. Here, we characterise the molecular structure of the CARP-receptor site in titin (UN2A) and its binding of CARP. We find that titin UN2A contains a central three-helix bundle fold (ca 45 residues in length) that is joined to N- and C-terminal flanking immunoglobulin domains by long, flexible linkers with partial helical content. CARP binds titin by engaging an α-hairpin in the three-helix fold of UN2A, the C-terminal linker sequence, and the BC loop in Ig81, which jointly form a broad binding interface. Mutagenesis showed that the CARP/N2A association withstands sequence variations in titin N2A and we use this information to evaluate 85 human single nucleotide variants. In addition, actin co-sedimentation, co-transfection in C2C12 cells, proteomics on heart lysates, and the mechanical response of CARP-soaked myofibrils imply that CARP induces the cross-linking of titin and actin myofilaments, thereby increasing myofibril stiffness. We conclude that CARP acts as a regulator of force output in the sarcomere that preserves muscle mechanical performance upon overload stress.

The Role of Cullin-RING Ligases in Striated Muscle Development, Function, and Disease.

The well-orchestrated turnover of proteins in cross-striated muscles is one of the fundamental processes required for muscle cell function and survival. Dysfunction of the intricate protein degradation machinery is often associated with development of cardiac and skeletal muscle myopathies. Most muscle proteins are degraded by the ubiquitin-proteasome system (UPS). The UPS involves a number of enzymes, including E3-ligases, which tightly control which protein substrates are marked for degradation by the proteasome. Recent data reveal that E3-ligases of the cullin family play more diverse and crucial roles in cross striated muscles than previously anticipated. This review highlights some of the findings on the multifaceted functions of cullin-RING E3-ligases, their substrate adapters, muscle protein substrates, and regulatory proteins, such as the Cop9 signalosome, for the development of cross striated muscles, and their roles in the etiology of myopathies.

The M-band: The underestimated part of the sarcomere.

The sarcomere is the basic unit of the myofibrils, which mediate skeletal and cardiac Muscle contraction. Two transverse structures, the Z-disc and the M-band, anchor the thin (actin and associated proteins) and thick (myosin and associated proteins) filaments to the elastic filament system composed of titin. A plethora of proteins are known to be integral or associated proteins of the Z-disc and its structural and signalling role in muscle is better understood, while the molecular constituents of the M-band and its function are less well defined. Evidence discussed here suggests that the M-band is important for managing force imbalances during active muscle contraction. Its molecular composition is fine-tuned, especially as far as the structural linkers encoded by members of the myomesin family are concerned and depends on the specific mechanical characteristics of each particular muscle fibre type. Muscle activity signals from the M-band to the nucleus and affects transcription of sarcomeric genes, especially via serum response factor (SRF). Due to its important role as shock absorber in contracting muscle, the M-band is also more and more recognised as a contributor to muscle disease.

Comparative analysis of obesity-related cardiometabolic and renal biomarkers in human plasma and serum.

The search for biomarkers associated with obesity-related diseases is ongoing, but it is not clear whether plasma and serum can be used interchangeably in this process. Here we used high-throughput screening to analyze 358 proteins and 76 lipids, selected because of their relevance to obesity-associated diseases, in plasma and serum from age- and sex-matched lean and obese humans. Most of the proteins/lipids had similar concentrations in plasma and serum, but a subset showed significant differences. Notably, a key marker of cardiovascular disease PAI-1 showed a difference in concentration between the obese and lean groups only in plasma. Furthermore, some biomarkers showed poor correlations between plasma and serum, including PCSK9, an important regulator of cholesterol homeostasis. Collectively, our results show that the choice of biofluid may impact study outcome when screening for obesity-related biomarkers and we identify several markers where this will be the case.

miR-486 is modulated by stretch and increases ventricular growth.

Perturbations in biomechanical stimuli during cardiac development contribute to congenital cardiac defects such as hypoplastic left heart syndrome (HLHS). This study sought to identify stretch-responsive pathways involved in cardiac development. miRNA-Seq identified miR-486 as being increased in cardiomyocytes exposed to cyclic stretch in vitro. The right ventricles (RVs) of patients with HLHS experienced increased stretch and had a trend toward higher miR-486 levels. Sheep RVs dilated from excessive pulmonary blood flow had 60% more miR-486 compared with control RVs. The left ventricles of newborn mice treated with miR-486 mimic were 16.9%-24.6% larger and displayed a 2.48-fold increase in cardiomyocyte proliferation. miR-486 treatment decreased FoxO1 and Smad signaling while increasing the protein levels of Stat1. Stat1 associated with Gata-4 and serum response factor (Srf), 2 key cardiac transcription factors with protein levels that increase in response to miR-486. This is the first report to our knowledge of a stretch-responsive miRNA that increases the growth of the ventricle in vivo.

Murine obscurin and Obsl1 have functionally redundant roles in sarcolemmal integrity, sarcoplasmic reticulum organization, and muscle metabolism.

Biological roles of obscurin and its close homolog Obsl1 (obscurin-like 1) have been enigmatic. While obscurin is highly expressed in striated muscles, Obsl1 is found ubiquitously. Accordingly, obscurin mutations have been linked to myopathies, whereas mutations in Obsl1 result in 3M-growth syndrome. To further study unique and redundant functions of these closely related proteins, we generated and characterized Obsl1 knockouts. Global Obsl1 knockouts are embryonically lethal. In contrast, skeletal muscle-specific Obsl1 knockouts show a benign phenotype similar to obscurin knockouts. Only deletion of both proteins and removal of their functional redundancy revealed their roles for sarcolemmal stability and sarcoplasmic reticulum organization. To gain unbiased insights into changes to the muscle proteome, we analyzed tibialis anterior and soleus muscles by mass spectrometry, uncovering additional changes to the muscle metabolism. Our analyses suggest that all obscurin protein family members play functions for muscle membrane systems.

Cullin-3 dependent deregulation of ACTN1 represents a new pathogenic mechanism in nemaline myopathy.

Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy and presence of nemaline bodies within myofibers. However, the understanding of underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40 and KLHL41, three substrate adaptors for the E3-ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for the disease development. Using Cullin-3 knockout mice, we identified accumulation of non-muscle alpha-Actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in KBTBD13 patients. Our data reveal that proper regulation of Cullin-3 activity and ACTN1 levels is essential for normal muscle and neuromuscular junction development. While ACTN1 is naturally downregulated during myogenesis, its overexpression in C2C12 myoblasts triggered defects in fusion, myogenesis and acetylcholine receptor clustering; features that we characterized in Cullin-3 deficient mice. Taken together, our data highlight the importance for Cullin-3 mediated degradation of ACTN1 for muscle development, and indicate a new pathomechanism for the etiology of myopathies seen in Cullin-3 knockout mice and nemaline myopathy patients.

PKC and PKN in heart disease.

The protein kinase C (PKC) and closely related protein kinase N (PKN) families of serine/threonine protein kinases play crucial cellular roles. Both kinases belong to the AGC subfamily of protein kinases that also include the cAMP dependent protein kinase (PKA), protein kinase B (PKB/AKT), protein kinase G (PKG) and the ribosomal protein S6 kinase (S6K). Involvement of PKC family members in heart disease has been well documented over the years, as their activity and levels are mis-regulated in several pathological heart conditions, such as ischemia, diabetic cardiomyopathy, as well as hypertrophic or dilated cardiomyopathy. This review focuses on the regulation of PKCs and PKNs in different pathological heart conditions and on the influences that PKC/PKN activation has on several physiological processes. In addition, we discuss mechanisms by which PKCs and the closely related PKNs are activated and turned-off in hearts, how they regulate cardiac specific downstream targets and pathways, and how their inhibition by small molecules is explored as new therapeutic target to treat cardiomyopathies and heart failure.

Mutant Muscle LIM Protein C58G causes cardiomyopathy through protein depletion.

Cysteine and glycine rich protein 3 (CSRP3) encodes Muscle LIM Protein (MLP), a well-established disease gene for Hypertrophic Cardiomyopathy (HCM). MLP, in contrast to the proteins encoded by the other recognised HCM disease genes, is non-sarcomeric, and has important signalling functions in cardiomyocytes. To gain insight into the disease mechanisms involved, we generated a knock-in mouse (KI) model, carrying the well documented HCM-causing CSRP3 mutation C58G. In vivo phenotyping of homozygous KI/KI mice revealed a robust cardiomyopathy phenotype with diastolic and systolic left ventricular dysfunction, which was supported by increased heart weight measurements. Transcriptome analysis by RNA-seq identified activation of pro-fibrotic signalling, induction of the fetal gene programme and activation of markers of hypertrophic signalling in these hearts. Further ex vivo analyses validated the activation of these pathways at transcript and protein level. Intriguingly, the abundance of MLP decreased in KI/KI mice by 80% and in KI/+ mice by 50%. Protein depletion was also observed in cellular studies for two further HCM-causing CSRP3 mutations (L44P and S54R/E55G). We show that MLP depletion is caused by proteasome action. Moreover, MLP C58G interacts with Bag3 and results in a proteotoxic response in the homozygous knock-in mice, as shown by induction of Bag3 and associated heat shock proteins. In conclusion, the newly generated mouse model provides insights into the underlying disease mechanisms of cardiomyopathy caused by mutations in the non-sarcomeric protein MLP. Furthermore, our cellular experiments suggest that protein depletion and proteasomal overload also play a role in other HCM-causing CSPR3 mutations that we investigated, indicating that reduced levels of functional MLP may be a common mechanism for HCM-causing CSPR3 mutations.

Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy.

Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2-associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure.

Cullin E3 Ligase Activity Is Required for Myoblast Differentiation.

The role of cullin E3-ubiquitin ligases for muscle homeostasis is best known during muscle atrophy, as the cullin-1 substrate adaptor atrogin-1 is among the most well-characterized muscle atrogins. We investigated whether cullin activity was also crucial during terminal myoblast differentiation and aggregation of acetylcholine receptors for the establishment of neuromuscular junctions in vitro. The activity of cullin E3-ligases is modulated through post-translational modification with the small ubiquitin-like modifier nedd8. Using either the Nae1 inhibitor MLN4924 (Pevonedistat) or siRNA against nedd8 in early or late stages of differentiation on C2C12 myoblasts, and primary satellite cells from mouse and human, we show that cullin E3-ligase activity is necessary for each step of the muscle cell differentiation program in vitro. We further investigate known transcriptional repressors for terminal muscle differentiation, namely ZBTB38, Bhlhe41, and Id1. Due to their identified roles for terminal muscle differentiation, we hypothesize that the accumulation of these potential cullin E3-ligase substrates may be partially responsible for the observed phenotype. MLN4924 is currently undergoing clinical trials in cancer patients, and our experiments highlight concerns on the homeostasis and regenerative capacity of muscles in these patients who often experience cachexia.