Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

Activation of the Aryl Hydrocarbon Receptor in Muscle Exacerbates Ischemic Pathology in Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) accelerates the development of atherosclerosis, decreases muscle function, and increases the risk of amputation or death in patients with peripheral artery disease (PAD). However, the mechanisms underlying this pathobiology are ill-defined. Recent work has indicated that tryptophan-derived uremic solutes, which are ligands for AHR (aryl hydrocarbon receptor), are associated with limb amputation in PAD. Herein, we examined the role of AHR activation in the myopathy of PAD and CKD. METHODS: AHR-related gene expression was evaluated in skeletal muscle obtained from mice and human PAD patients with and without CKD. AHRmKO (skeletal muscle-specific AHR knockout) mice with and without CKD were subjected to femoral artery ligation, and a battery of assessments were performed to evaluate vascular, muscle, and mitochondrial health. Single-nuclei RNA sequencing was performed to explore intercellular communication. Expression of the constitutively active AHR was used to isolate the role of AHR in mice without CKD. RESULTS: PAD patients and mice with CKD displayed significantly higher mRNA expression of classical AHR-dependent genes (Cyp1a1, Cyp1b1, and Aldh3a1) when compared with either muscle from the PAD condition with normal renal function (P<0.05 for all 3 genes) or nonischemic controls. AHRmKO significantly improved limb perfusion recovery and arteriogenesis, preserved vasculogenic paracrine signaling from myofibers, increased muscle mass and strength, as well as enhanced mitochondrial function in an experimental model of PAD/CKD. Moreover, viral-mediated skeletal muscle-specific expression of a constitutively active AHR in mice with normal kidney function exacerbated the ischemic myopathy evidenced by smaller muscle masses, reduced contractile function, histopathology, altered vasculogenic signaling, and lower mitochondrial respiratory function. CONCLUSIONS: These findings establish AHR activation in muscle as a pivotal regulator of the ischemic limb pathology in CKD. Further, the totality of the results provides support for testing of clinical interventions that diminish AHR signaling in these conditions.

Chronic activation of the aryl hydrocarbon receptor in muscle exacerbates ischemic pathology in chronic kidney disease.

Chronic kidney disease (CKD) accelerates the development of atherosclerosis, decreases muscle function, and increases the risk of amputation or death in patients with peripheral artery disease (PAD). However, the cellular and physiological mechanisms underlying this pathobiology are ill-defined. Recent work has indicated that tryptophan-derived uremic toxins, many of which are ligands for the aryl hydrocarbon receptor (AHR), are associated with adverse limb outcomes in PAD. We hypothesized that chronic AHR activation, driven by the accumulation of tryptophan-derived uremic metabolites, may mediate the myopathic condition in the presence of CKD and PAD. Both PAD patients with CKD and mice with CKD subjected to femoral artery ligation (FAL) displayed significantly higher mRNA expression of classical AHR-dependent genes ( Cyp1a1 , Cyp1b1 , and Aldh3a1 ) when compared to either muscle from the PAD condition with normal renal function ( P <0.05 for all three genes) or non-ischemic controls. Skeletal-muscle-specific AHR deletion in mice (AHR mKO ) significantly improved limb muscle perfusion recovery and arteriogenesis, preserved vasculogenic paracrine signaling from myofibers, increased muscle mass and contractile function, as well as enhanced mitochondrial oxidative phosphorylation and respiratory capacity in an experimental model of PAD/CKD. Moreover, viral-mediated skeletal muscle-specific expression of a constitutively active AHR in mice with normal kidney function exacerbated the ischemic myopathy evidenced by smaller muscle masses, reduced contractile function, histopathology, altered vasculogenic signaling, and lower mitochondrial respiratory function. These findings establish chronic AHR activation in muscle as a pivotal regulator of the ischemic limb pathology in PAD. Further, the totality of the results provide support for testing of clinical interventions that diminish AHR signaling in these conditions.

Skeletal muscle Nox4 knockout prevents and Nox2 knockout blunts loss of maximal diaphragm force in mice with heart failure with reduced ejection fraction.

Patients with heart failure with reduced ejection fraction (HFrEF) experience diaphragm weakness that contributes to the primary disease symptoms of fatigue, dyspnea, and exercise intolerance. Weakness in the diaphragm is related to excessive production of reactive oxygen species (ROS), but the exact source of ROS remains unknown. NAD(P)H Oxidases (Nox), particularly the Nox2 and 4 isoforms, are important sources of ROS within skeletal muscle that contribute to optimal cell function. There are reports of increased Nox activity in the diaphragm of patients and animal models of HFrEF, implicating these complexes as possible sources of diaphragm dysfunction in HFrEF. To investigate the role of these proteins on diaphragm weakness in HFrEF, we generated inducible skeletal muscle specific knockouts of Nox2 or Nox4 using the Cre-Lox system and assessed diaphragm function in a mouse model of HFrEF induced by myocardial infarction. Diaphragm maximal specific force measured in vitro was depressed by ∼20% with HFrEF. Skeletal muscle knockout of Nox4 provided full protection against the loss of maximal force (p < 0.01), while the knockout of Nox2 provided partial protection (7% depression, p < 0.01). Knockout of Nox2 from skeletal myofibers improved survival from 50 to 80% following myocardial infarction (p = 0.026). Our findings show an important role for skeletal muscle NAD(P)H Oxidases contributing to loss of diaphragm maximal force in HFrEF, along with systemic pathophysiological responses following myocardial infarction.

Cardiac and respiratory muscle responses to dietary N-acetylcysteine in rats consuming a high-saturated fat, high-sucrose diet.

NEW FINDINGS: What is the central question of this study? This study addresses whether a high-fat, high-sucrose diet causes cardiac and diaphragm muscle abnormalities in male rats and whether supplementation with the antioxidant N-acetylcysteine reverses diet-induced dysfunction. What is the main finding and its importance? N-Acetylcysteine attenuated the effects of high-fat, high-sucrose diet on markers of cardiac hypertrophy and diastolic dysfunction, but neither high-fat, high-sucrose diet nor N-acetylcysteine affected the diaphragm. These results support the use of N-acetylcysteine to attenuate cardiovascular dysfunction induced by a 'Western' diet. ABSTRACT: Individuals with overweight or obesity display respiratory and cardiovascular dysfunction, and oxidative stress is a causative factor in the general aetiology of obesity and of skeletal and cardiac muscle pathology. Thus, this preclinical study aimed to define diaphragmatic and cardiac morphological and functional alterations in response to an obesogenic diet in rats and the therapeutic potential of an antioxidant supplement, N-acetylcysteine (NAC). Young male Wistar rats consumed ad libitum a 'lean' or high-saturated fat, high-sucrose (HFHS) diet for ∼22 weeks and were randomized to control or NAC (2 mg/ml in the drinking water) for the last 8 weeks of the dietary intervention. We then evaluated diaphragmatic and cardiac morphology and function. Neither HFHS diet nor NAC supplementation affected diaphragm-specific force, peak power or morphology. Right ventricular weight normalized to estimated body surface area, left ventricular fractional shortening and posterior wall maximal shortening velocity were higher in HFHS compared with lean control animals and not restored by NAC. In HFHS rats, the elevated deceleration rate of early transmitral diastolic velocity was prevented by NAC. Our data showed that the HFHS diet did not compromise diaphragmatic muscle morphology or in vitro function, suggesting other possible contributors to breathing abnormalities in obesity (e.g., abnormalities of neuromuscular transmission). However, the HFHS diet resulted in cardiac functional and morphological changes suggestive of hypercontractility and diastolic dysfunction. Supplementation with NAC did not affect diaphragm morphology or function but attenuated some of the cardiac abnormalities in the rats receiving the HFHS diet.

Reversible Thiol Oxidation Increases Mitochondrial Electron Transport Complex Enzyme Activity but Not Respiration in Cardiomyocytes from Patients with End-Stage Heart Failure.

Cardiomyocyte dysfunction in patients with end-stage heart failure with reduced ejection fraction (HFrEF) stems from mitochondrial dysfunction, which contributes to an energetic crisis. Mitochondrial dysfunction reportedly relates to increased markers of oxidative stress, but the impact of reversible thiol oxidation on myocardial mitochondrial function in patients with HFrEF has not been investigated. In the present study, we assessed mitochondrial function in ventricular biopsies from patients with end-stage HFrEF in the presence and absence of the thiol-reducing agent dithiothreitol (DTT). Isolated mitochondria exposed to DTT had increased enzyme activity of complexes I (p = 0.009) and III (p = 0.018) of the electron transport system, while complexes II (p = 0.630) and IV (p = 0.926) showed no changes. However, increased enzyme activity did not carry over to measurements of mitochondrial respiration in permeabilized bundles. Oxidative phosphorylation conductance (p = 0.439), maximal respiration (p = 0.312), and ADP sensitivity (p = 0.514) were unchanged by 5 mM DTT treatment. These results indicate that mitochondrial function can be modulated through reversible thiol oxidation, but other components of mitochondrial energy transfer are rate limiting in end-stage HFrEF. Optimal therapies to normalize cardiac mitochondrial respiration in patients with end-stage HFrEF may benefit from interventions to reverse thiol oxidation, which limits complex I and III activities.

Nitric oxide and skeletal muscle contractile function.

Nitric oxide (NO) is complex modulator of skeletal muscle contractile function, capable of increasing or decreasing force and power output depending on multiple factors. This review explores the effects and potential mechanisms for modulation of skeletal muscle contractile function by NO, from pharmacological agents in isolated muscle preparations to dietary nitrate supplementation in humans and animals. Pharmacological manipulation in vitro suggests that NO signaling diminishes submaximal isometric force, whereas dietary manipulation in vivo suggest that NO enhances submaximal force. The bases for these different responses are unknown but could reflect dose-dependent effects. Maximal isometric force is unaffected by physiologically relevant levels of NO, which do not induce overt protein oxidation. Pharmacological and dietary manipulation of NO signaling enhances the maximal rate of isometric force development, unloaded shortening velocity, and peak power. We hypothesize that these effects are mediated by post-translational modifications of myofibrillar proteins that modulate thick filament regulation of contraction (e.g., mechanosensing and strain-dependence of cross-bridge kinetics). NO effects on contractile function appear to have some level of fiber type and sex-specificity. The mechanisms behind NO-mediated changes in skeletal muscle function need to be explored through proteomics analysis and advanced biophysical assays to advance the development of small molecules and open intriguing therapeutic and ergogenic possibilities for aging, disease, and athletic performance.

Skeletal myopathy in a rat model of postmenopausal heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) accounts for ∼50% of all patients with heart failure and frequently affects postmenopausal women. The HFpEF condition is phenotype-specific, with skeletal myopathy that is crucial for disease development and progression. However, most of the current preclinical models of HFpEF have not addressed the postmenopausal phenotype. We sought to advance a rodent model of postmenopausal HFpEF and examine skeletal muscle abnormalities therein. Female, ovariectomized, spontaneously hypertensive rats (SHRs) were fed a high-fat, high-sucrose diet to induce HFpEF. Controls were female sham-operated Wistar-Kyoto rats on a lean diet. In a complementary, longer-term cohort, controls were female sham-operated SHRs on a lean diet to evaluate the effect of strain difference in the model. Our model developed key features of HFpEF that included increased body weight, glucose intolerance, hypertension, cardiac hypertrophy, diastolic dysfunction, exercise intolerance, and elevated plasma cytokines. In limb skeletal muscle, HFpEF decreased specific force by 15%-30% (P < 0.05) and maximal mitochondrial respiration by 40%-55% (P < 0.05), increased oxidized glutathione by approximately twofold (P < 0.05), and tended to increase mitochondrial H2O2 emission (P = 0.10). Muscle fiber cross-sectional area, markers of mitochondrial content, and indices of capillarity were not different between control and HFpEF in our short-term cohort. Overall, our preclinical model of postmenopausal HFpEF recapitulates several key features of the disease. This new model reveals contractile and mitochondrial dysfunction and redox imbalance that are potential contributors to abnormal metabolism, exercise intolerance, and diminished quality of life in patients with postmenopausal HFpEF.NEW & NOTEWORTHY Heart failure with preserved ejection fraction (HFpEF) is a condition with phenotype-specific features highly prevalent in postmenopausal women and skeletal myopathy contributing to disease development and progression. We advanced a rat model of postmenopausal HFpEF with key cardiovascular and systemic features of the disease. Our study shows that the skeletal myopathy of postmenopausal HFpEF includes loss of limb muscle-specific force independent of atrophy, mitochondrial dysfunction, and oxidized shift in redox balance.

Nox4 Knockout Does Not Prevent Diaphragm Atrophy, Contractile Dysfunction, or Mitochondrial Maladaptation in the Early Phase Post-Myocardial Infarction in Mice.

BACKGROUND/AIMS: Diaphragm dysfunction with increased reactive oxygen species (ROS) occurs within 72 hrs post-myocardial infarction (MI) in mice and may contribute to loss of inspiratory maximal pressure and endurance in patients. METHODS: We used wild-type (WT) and whole-body Nox4 knockout (Nox4KO) mice to measure diaphragm bundle force in vitro with a force transducer, mitochondrial respiration in isolated fiber bundles with an O2 sensor, mitochondrial ROS by fluorescence, mRNA (RT-PCR) and protein (immunoblot), and fiber size by histology 72 hrs post-MI. RESULTS: MI decreased diaphragm fiber cross-sectional area (CSA) (~15%, p = 0.015) and maximal specific force (10%, p = 0.005), and increased actin carbonylation (5-10%, p = 0.007) in both WT and Nox4KO. Interestingly, MI did not affect diaphragm mRNA abundance of MAFbx/atrogin-1 and MuRF-1 but Nox4KO decreased it by 20-50% (p < 0.01). Regarding the mitochondria, MI and Nox4KO decreased the protein abundance of citrate synthase and subunits of electron transport system (ETS) complexes and increased mitochondrial O2 flux (JO2) and H2O2 emission (JH2O2) normalized to citrate synthase. Mitochondrial electron leak (JH2O2/JO2) in the presence of ADP was lower in Nox4KO and not changed by MI. CONCLUSION: Our study shows that the early phase post-MI causes diaphragm atrophy, contractile dysfunction, sarcomeric actin oxidation, and decreases citrate synthase and subunits of mitochondrial ETS complexes. These factors are potential causes of loss of inspiratory muscle strength and endurance in patients, which likely contribute to the pathophysiology in the early phase post-MI. Whole-body Nox4KO did not prevent the diaphragm abnormalities induced 72 hrs post-MI, suggesting that systemic pharmacological inhibition of Nox4 will not benefit patients in the early phase post-MI.

The impact of hindlimb disuse on sepsis-induced myopathy in mice.

Sepsis induces a myopathy characterized by loss of muscle mass and weakness. Septic patients undergo prolonged periods of limb muscle disuse due to bed rest. The contribution of limb muscle disuse to the myopathy phenotype remains poorly described. To characterize sepsis-induced myopathy with hindlimb disuse, we combined the classic sepsis model via cecal ligation and puncture (CLP) with the disuse model of hindlimb suspension (HLS) in mice. Male C57bl/6j mice underwent CLP or SHAM surgeries. Four days after surgeries, mice underwent HLS or normal ambulation (NA) for 7 days. Soleus (SOL) and extensor digitorum longus (EDL) were dissected for in vitro muscle mechanics, morphological, and histological assessments. In SOL muscles, both CLP+NA and SHAM+HLS conditions elicited ~20% reduction in specific force (p < 0.05). When combined, CLP+HLS elicited ~35% decrease in specific force (p < 0.05). Loss of maximal specific force (~8%) was evident in EDL muscles only in CLP+HLS mice (p < 0.05). CLP+HLS reduced muscle fiber cross-sectional area (CSA) and mass in SOL (p < 0.05). In EDL muscles, CLP+HLS decreased absolute mass to a smaller extent (p < 0.05) with no changes in CSA. Immunohistochemistry revealed substantial myeloid cell infiltration (CD68+) in SOL, but not in EDL muscles, of CLP+HLS mice (p < 0.05). Combining CLP with HLS is a feasible model to study sepsis-induced myopathy in mice. Hindlimb disuse combined with sepsis induced muscle dysfunction and immune cell infiltration in a muscle dependent manner. These findings highlight the importance of rehabilitative interventions in septic hosts to prevent muscle disuse and help attenuate the myopathy.