AIMS:Skeletal muscle energetic augmentation might be a mechanism via which intravenous iron improves symptoms in heart failure, but no direct measurement of intrinsic mitochondrial function has been performed to support this notion. This molecular substudy of the FERRIC-HF II trial tested the hypothesis that ferric derisomaltose (FDI) would improve electron transport chain activity, given its high dependence on iron-sulfur clusters which facilitate electron transfer during oxidative phosphorylation. METHODS AND RESULTS:Vastus lateralis skeletal muscle biopsies were taken before and 2 weeks after randomization. Mitochondrial complex I, II, and I&II respiration were quantified with respirometry of permeabilized fresh skeletal muscle biopsies. Net respiratory capacities, reflecting respiration that is truly available for adenosine triphosphate generation, were calculated by subtracting non-phosphorylating LEAK respiration. Complex I-V and myoglobin protein levels, and skeletal muscle fibre type composition were assayed. Patients randomised to FDI (n = 21) or placebo (n = 19) were similar (age 66 ± 13 years, 73% men, left ventricular ejection fraction 37 ± 8%, 48% New York Heart Association class III, 50% diabetic). After 2 weeks, total complex I-linked respiration (0.33 [interquartile range 0.24-0.37] vs. 0.19 [0.06-0.27] nmol/min/mg, p = 0.03) and net complex I-linked respiration (0.21 [0.16-0.24] vs. 0.11 [0.04-0.16] nmol/min/mg, p = 0.01) were higher in patients allocated to FDI. There was no intergroup difference in other respiratory states, in mitochondrial abundance as reflected by complex I-V protein levels, and in skeletal muscle myoglobin and oxidative fibre type content. CONCLUSIONS:Iron repletion induces an early, selective, and potentially direct enhancement of mitochondrial complex I-dependent respiration in the skeletal muscle of heart failure patients. This could be harnessed to optimize repletion protocols to maximize patient benefits.

The lamin A precursor, prelamin A, requires extensive processing to yield mature lamin A and effect its primary function as a structural filament of the nucleoskeleton. When processing is perturbed, nuclear accumulation of prelamin A is toxic and causes laminopathic diseases such as Hutchinson-Gilford progeria syndrome and cardiomyopathy. However, the physiological role of prelamin A is largely unknown and we sought to identify novel insights about this. Using rodent heart tissue, primary cells and the C2C12 model of myofibrillogenesis, we investigated the expression and localization patterns of prelamin A in heart and skeletal muscle cells. We found that endogenous prelamin A was detectable in mouse heart localized to the sarcomere in both adult mouse heart and isolated neonatal rat cardiomyocytes. We investigated the regulation of prelamin A in C2C12 myofibrillogenesis and found it was dynamically regulated and organized into striations upon myofibril formation, colocalizing with the Z-disc protein α-actinin. These data provide evidence that prelamin A is a component of the sarcomere, underpinning a physiological purpose for unprocessed prelamin A. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

The thermoneutral zone (TNZ) defines the range of ambient temperatures at which resting metabolic rate (MR) is at a minimum. While the TNZ lower limit has been characterized, it is still unclear whether there is an upper limit, that is, beyond which MR during rest increases, and if so, what physiological upregulations explain this. We take the first step to fill this knowledge gap by measuring MR and multiple physiological variables in participants exposed to ambient heat stress while resting. Thirteen participants were exposed for an hour to 28℃‐50% relative humidity (RH) air, and both 40 and 50℃ each in 25% RH and humid (50% RH) conditions. Core and skin temperatures, blood pressure, sweat‐, heart‐, and breathing‐rate, minute ventilation, and movement levels were recorded throughout each condition. MR increased 35% (p = .015) during exposure to 40℃‐25% RH compared to baseline and a further 13% (p = .000) at in 50℃‐50%RH. This was not explained by increased fidgeting (p = .26), suggesting physiological upregulation. However, while greater heat stress invoked increases in heart rate (64%, p = .000), minute ventilation (78%, p = .000), and sweat rate (74%. p = .000) when comparing 50℃‐50% RH with baseline, the exact size of their relative energy cost is unclear and, therefore, so is their contribution to this increase in MR. Our study shows clear evidence that resting MR increases in humans at high temperature—there is a metabolic upper critical temperature, at least as low as 40℃. Further studies should pinpoint this value and fully explain this increased MR.

Abstract ΔR4-R23/ΔCT micro-dystrophin (μDys) is a miniaturized version of dystrophin currently evaluated in a Duchenne muscular dystrophy (DMD) gene therapy trial to treat skeletal and cardiac muscle disease. In pre-clinical studies, μDys efficiently rescues cardiac histopathology, but only partially normalizes cardiac function. To gain insights into factors that may impact the cardiac therapeutic efficacy of μDys, we compared by mass spectrometry the composition of purified dystrophin and μDys protein complexes in the mouse heart. We report that compared to dystrophin, μDys has altered associations with α1- and β2-syntrophins, as well as cavins, a group of caveolae-associated signaling proteins. In particular, we found that membrane localization of cavin-1 and cavin-4 in cardiomyocytes requires dystrophin and is profoundly disrupted in the heart of mdx5cv mice, a model of DMD. Following cardiac stress/damage, membrane-associated cavin-4 recruits the signaling molecule ERK to caveolae, which activates key cardio-protective responses. Evaluation of ERK signaling revealed a profound inhibition, below physiological baseline, in the mdx5cv mouse heart. Expression of μDys in mdx5cv mice prevented the development of cardiac histopathology but did not rescue membrane localization of cavins nor did it normalize ERK signaling. Our study provides the first comparative analysis of purified protein complexes assembled in vivo by full-length dystrophin and a therapeutic micro-dystrophin construct. This has revealed disruptions in cavins and ERK signaling that may contribute to DMD cardiomyopathy. This new knowledge is important for ongoing efforts to prevent and treat heart disease in DMD patients.

Background Patients with repair of tetralogy of Fallot (rToF) who are approaching adulthood often exhibit pulmonary valve regurgitation, leading to right ventricle (RV) dilatation and dysfunction. The regurgitation can be corrected by pulmonary valve replacement (PVR), but the optimal surgical timing remains under debate, mainly because of the poorly understood nature of RV remodeling in patients with rToF. The goal of this study was to probe for pathologic molecular, cellular, and tissue changes in the myocardium of patients with rToF at the time of PVR. Methods and Results We measured contractile function of permeabilized myocytes, collagen content of tissue samples, and the expression of mRNA and selected proteins in RV tissue samples from patients with rToF undergoing PVR for severe pulmonary valve regurgitation. The data were compared with nondiseased RV tissue from unused donor hearts. Contractile performance and passive stiffness of the myofilaments in permeabilized myocytes were similar in rToF‐PVR and RV donor samples, as was collagen content and cross‐linking. The patients with rToF undergoing PVR had enhanced mRNA expression of genes associated with connective tissue diseases and tissue remodeling, including the small leucine‐rich proteoglycans ASPN (asporin), LUM (lumican), and OGN (osteoglycin), although their protein levels were not significantly increased. Conclusions RV myofilaments from patients with rToF undergoing PVR showed no functional impairment, but the changes in extracellular matrix gene expression may indicate the early stages of remodeling. Our study found no evidence of major damage at the cellular and tissue levels in the RV of patients with rToF who underwent PVR according to current clinical criteria.

Cardiomyopathies are progressive diseases of heart muscle often caused by mutations in genes encoding sarcomeric, cytoskeletal and nucleoskeletal proteins though in many cases the cause of disease is not identified. Whilst nucleus hypertrophy has been described, it is not known whether nucleus shape changes are a general feature of cardiomyopathy. Due to the rod-shaped nature of cardiomyocytes and their elliptical nuclei we hypothesised that orientation of analysis would be an important determinant of any changes observed between patients exhibiting primarily unexplained cardiomyopathy and control samples from non-failing donors. To investigate this we performed image analysis of cardiomyocyte nuclei in myocardial cryosections from a cohort of cardiomyopathy patients. We discovered that circularity, solidity and aspect ratio were sensitive to orientation of the myocardium and that in the transverse plane only circularity was reduced in cardiomyocyte nuclei of cardiomyopathy patients. These findings show that orientation dependent changes in nucleus shape may be a property of cardiomyopathy and with appropriate follow up studies, may prove to have mechanistic and diagnostic value.

Regulation of the genome is viewed through the prism of gene expression, DNA replication and DNA repair as controlled through transcription, chromatin compartmentalisation and recruitment of repair factors by enzymes such as DNA polymerases, ligases, acetylases, methylases and cyclin-dependent kinases. However, recent advances in the field of muscle cell physiology have also shown a compelling role for ‘outside-in’ biophysical control of genomic material through mechanotransduction. The crucial hub that transduces these biophysical signals is called the Linker of Nucleoskeleton and Cytoskeleton (LINC). This complex is embedded across the nuclear envelope, which separates the nucleus from the cytoplasm. How the LINC complex operates to mechanically regulate the many functions of DNA is becoming increasingly clear, and recent advances have provided exciting insight into how this occurs in cells from mechanically activated tissues such as skeletal and cardiac muscle. Nevertheless, there are still some notable shortcomings in our understanding of these processes and resolving these will likely help us understand how muscle diseases manifest at the level of the genome.

Cardiomyopathies are complex heart muscle diseases that can be inherited or acquired. Dilated cardiomyopathy can result from mutations in LMNA, encoding the nuclear intermediate filament proteins lamin A/C. Some LMNA mutations lead to accumulation of the lamin A precursor, prelamin A, which is disease causing in a number of tissues, yet its impact upon the heart is unknown. Here, we discovered myocardial prelamin A accumulation occurred in a case of dilated cardiomyopathy, and we show that a potentially novel mouse model of cardiac-specific prelamin A accumulation exhibited a phenotype consistent with inflammatory cardiomyopathy, which we observed to be similar to HIV-associated cardiomyopathy, an acquired disease state. Numerous HIV protease therapies are known to inhibit ZMPSTE24, the enzyme responsible for prelamin A processing, and we confirmed that accumulation of prelamin A occurred in HIV+ patient cardiac biopsies. These findings (a) confirm a unifying pathological role for prelamin A common to genetic and acquired cardiomyopathies; (b) have implications for the management of HIV patients with cardiac disease, suggesting protease inhibitors should be replaced with alternative therapies (i.e., nonnucleoside reverse transcriptase inhibitors); and (c) suggest that targeting inflammation may be a useful treatment strategy for certain forms of inherited cardiomyopathy.

Participation in ultra-endurance cycling events, such as the Transcontinental Race, is increasing. These extremely demanding races provide a unique opportunity for field observation of the limits of human endurance physiology and, importantly, when these limits might be exceeded and cross over into pathology. The heart is of special interest in this field, and previous data suggest that 'reverse drift' of heart rate occurs as a product of time and load in races of 24-48 h, whereas transient structural abnormalities have been observed upon completion of running ultramarathons. Here, we report a unique case of a male cyclist racing in the Transcontinental Race over an extended period of 14 days characterized by extreme workloads and a low quantity and quality of sleep. The heart rate response was dynamic over the course of the race and defined by a U-shaped quadratic relationship. A larger scale study is required to determine the relevance of this information to the ultra-endurance cycling community.

Cardiomyopathies are complex heart muscle diseases that can be inherited e.g. dilated cardiomyopathy resulting from LMNA gene mutations, or acquired, e.g. cardiomyopathy associated with HIV. In both cases the lamin A precursor, prelamin A, may play a central role: mutations in LMNA and certain HIV protease inhibitors acting via the enzyme ZMPSTE24 both inhibit prelamin A processing. Firstly, we show that myocardial prelamin A accumulation occurs in both these cardiomyopathies in patients. Secondly, we developed a novel mouse model of cardiac specific prelamin A accumulation which mimicked tissue and molecular features of HIV associated cardiomyopathy, including inflammation. These findings: (1) confirm a central pathological role of prelamin A common to genetic and acquired cardiomyopathies; (2) have implications for the management of HIV patients with cardiac disease in whom protease inhibitors with low/no binding to ZMPSTE24 may be preferred; and (3) suggest that targeting inflammation may be a useful treatment strategy for some forms of inherited cardiomyopathy.

In this issue, Wang et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201708137) show that disruption to different mechanical domains of muscle cells converge at the linker of nucleoskeleton to cytoskeleton complex to affect DNA endoreplication potentially via barrier to autointegration factor-mediated epigenetic mechanisms.

The nuclear lamina is a critical structural domain for the maintenance of genomic stability and whole-cell mechanics. Mutations in the LMNA gene, which encodes nuclear A-type lamins lead to the disruption of these key cellular functions, resulting in a number of devastating diseases known as laminopathies. Cardiomyopathy is a common laminopathy and is highly penetrant with poor prognosis. To date, cell mechanical instability and dysregulation of gene expression have been proposed as the main mechanisms driving cardiac dysfunction, and indeed discoveries in these areas have provided some promising leads in terms of therapeutics. However, important questions remain unanswered regarding the role of lamin A dysfunction in the heart, including a potential role for the toxicity of lamin A precursors in LMNA cardiomyopathy, which has yet to be rigorously investigated.

Introduction Mutations in the LMNA gene, which encodes the nuclear intermediate filament proteins lamins A and C, lead to a number of premature ageing syndromes, including Hutchinson Gilford Progeria syndrome (HGPS) and Emery Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). Some causal mutations disrupt lamin A processing, resulting in the accumulation of the lamin A precursor, prelamin A, however it is not clear to what extent accumulated prelamin A contributes to DCM. Here we identify DCM patients with cardiomyocyte accumulation of prelamin A and explore the impact of prelamin A accumulation in vivo. Methods We generated a novel line of targeted transgenic mice that accumulate prelamin A specifically in cardiomyocytes (PLA Tg mice), by expressing an uncleavable form of prelamin A, driven by Cre expression from the myosin light chain 2 ventricular (MLC2v) promoter. Results Immunofluorescence staining of human DCM biopsies showed the presence of nuclear prelamin A in cardiomyocytes. PLA Tg mice were born without any obvious phenotype but manifested retarded growth by 3 weeks of age and succumbed to heart failure at ˜5 weeks. At 4 weeks, MRI showed a marked dilatation of the cardiac chambers and a decline in cardiac function in vivo. Ejection fraction (EF) was substantially depressed in PLA Tg micecompared with wildtype indicating DCM and heart failure. Cardiac histology showed marked cardiomyocyte disarray and profound fibrosis. Biochemical and microscopic analyses indicated disruption of the linkers of nucleoskeleton to cytoskeleton complex and perinuclear intermediate filament network and was supported by electron micrographs showing nuclear morphology defects. Myocardial infiltration of CD45 positive cells coincided with the expression of γ-H2AX, phosphorylated ATM and increased NF-κB signalling, which suggested an inflammatory response initiated by DNA damage. This may be related to the senescence associated secretory phenotype (SASP) as senescence associated β-galactosidase was also expressed in PLA Tg myocardium. Conclusion We have demonstrated prelamin A accumulation in the nuclei of human DCM biopsies and show that overexpression of prelamin A in a transgenic mouse model leads to an early decline in cardiac function and premature myocardial senescence.

Ageing is a potent risk factor for cardiovascular (CV) disease. Mutations in the LMNA gene, which encodes the nuclear intermediate filament proteins lamins A & C, lead to a number of premature ageing syndromes, e.g. Emery Dreifuss muscular dystrophy (EDMD), which can lead to dilated cardiomyopathy (DCM) and heart failure. Some of these mutations affect lamin A processing, resulting in the accumulation of the lamin A precursor, prelamin A. Importantly, recent studies have shown that the mechanisms of premature ageing observed in patients with lamin A mutations may also occur during normal ageing processes. This led us to postulate that accumulation of prelamin A in the heart may mimic ‘age-related’ cardiac dysfunction. To test this, we generated a novel line of targeted transgenic mice that accumulate prelamin A specifically in cardiomyocytes, by expressing a modified prelamin A gene driven by the myosin light chain 2 ventricular (MLC2v) promoter. These mice were born without any obvious phenotype but manifested retarded growth by 3 weeks of age and had a significantly attenuated lifespan (∼5 weeks) with the cause of death appearing to be heart failure. Their failure to thrive was also underscored by a muscular dystrophy like appearance. At 4 weeks age, echocardiography showed a marked dilatation of the cardiac chambers and a decline in cardiac function in vivo, e.g. ejection fraction (EF) was substantially depressed in transgenic mice (20.9±3.6%) compared with wildtype (56.7±11.2%, P=0.01), indicating DCM and heart failure. Cardiac histology showed marked cardiomyocyte disarray, profound fibrosis (Picro-sirius Red staining), and expression of senescence markers (senescence-associated beta-galactosidase). Analyses of molecular events indicated severe disruption of the LINC complex and perinuclear intermediate filament network as a number of components (SUN 2, Nesprin 2, Desmin) underwent significant expression changes and/or were mislocalised. Additionally, immunohistochemistry and western blot for CD45 and CD68 confirmed increased numbers of inflammatory cells in hearts overexpressing prelamin A. In conclusion, prelamin A accumulation in cardiomyocytes causes a pathogenic response leading to decline in cardiac function and, ultimately, death. Further investigation aims to establish the precise mechanisms underpinning the response of the heart to accumulation of prelamin A.

Summary Rnd proteins are atypical Rho family proteins that do not hydrolyse GTP and are instead regulated by expression levels and post-translational modifications. Rnd1 and Rnd3/RhoE induce loss of actin stress fibres and cell rounding in multiple cell types, whereas responses to Rnd2 are more variable. Here we report the responses of endothelial cells to Rnd proteins. Rnd3 induces a very transient decrease in stress fibres but subsequently stimulates a strong increase in stress fibres, in contrast to the reduction observed in other cell types. Rnd2 also increases stress fibres whereas Rnd1 induces a loss of stress fibres and weakening of cell–cell junctions. Rnd3 does not act through any of its known signalling partners and does not need to associate with membranes to increase stress fibres. Instead, it acts by increasing RhoB expression, which is then required for Rnd3-induced stress fibre assembly. Rnd2 also increases RhoB levels. These data indicate that the cytoskeletal response to Rnd3 expression is dependent on cell type and context, and identify regulation of RhoB as a new mechanism for Rnd proteins to affect the actin cytoskeleton.