Assessing equity in the uptake of remote foot temperature monitoring in a large integrated US healthcare system.

OBJECTIVE: We assessed equity in the uptake of remote foot temperature monitoring (RTM) for amputation prevention throughout a large, integrated US healthcare system between 2019 and 2021, including comparisons across facilities and between patients enrolled and eligible patients not enrolled in RTM focusing on the Reach and Adoption dimensions of the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework. MATERIAL AND METHODS: To assess whether there was equitable use of RTM across facilities, we examined distributions of patient demographic, geographic, and facility characteristics across facility RTM use categories (e.g., no RTM use, and low, moderate, and high RTM use) among all eligible patients (n = 46,294). Second, to understand whether, among facilities using RTM, there was equitable enrollment of patients in RTM, we compared characteristics of patients enrolled in RTM (n = 1066) relative to a group of eligible patients not enrolled in RTM (n = 27,166) using logistic regression and including all covariates. RESULTS: RTM use increased substantially from an average of 11 patients per month to over 40 patients per month between 2019 and 2021. High-use RTM facilities had higher complexity and a lower ratio of patients per podiatrist but did not have consistent evidence of better footcare process measures. Among facilities offering RTM, enrollment varied by age, was inversely associated with Black race (vs. white), low income, living far from specialty care, and being in the highest quartiles of telehealth use prior to enrollment. Enrollment was positively associated with having osteomyelitis, Charcot foot, a partial foot amputation, BMI≥30 kg/m2, and high outpatient utilization. CONCLUSIONS: RTM growth was concentrated in a small number of higher-resourced facilities, with evidence of lower enrollment among those who were Black and lived farther from specialty care. Future studies are needed to identify and address barriers to uptake of new interventions like RTM to prevent exacerbating existing ulceration and amputation disparities.

Evidence-Based Opioid Education That Reduces Prescribing: The 10 Principles of Opioid Prescribing in Foot and Ankle Surgery.

Over half of opioid misusers last obtained access to opioids via a friend or relative, a problematic reflection of the opioid reservoir phenomenon, which results from an unused backlog of excess prescription opioids that are typically stored in the American home. We aim to determine if a voluntary educational intervention containing standard opioid and nonopioid analgesic prescribing ranges for common surgeries is effective in altering postoperative prescribing practice. We utilized a mixed methods approach and sent out a questionnaire to American podiatric physicians, including residents (baseline group A), via email in early 2020 for baseline data; then, we interviewed foot and ankle surgeons and the primary themes of these semistructured interviews informed us to target residents for an educational intervention. We repeated the survey 3 years later in summer 2022 (preintervention group B). We created an opioid guide and emailed it to residents in fall 2022. Another repeat survey was done in spring 2023 (postintervention group C). We used the Mann-Whitney U test to examine differences between the groups among their reported postoperative opioid quantities for a first metatarsal osteotomy surgical scenario. Groups A, B, and C had 60, 100, and 99 residents, respectively. There was no significant difference (p = .9873) between baseline group A and preintervention group B. There was a difference (p < .0001; -5 median) between preintervention group B and postintervention group C (same residency year). In postintervention group C, a majority (91/99) reported viewing the guide at least once, and the number of residents that reported supplementing with NSAIDs also doubled compared to preintervention group B. This novel opioid educational intervention resulted in meaningful change in self-reported postoperative prescribing behavior among residents.

Evaluation of the Effectiveness of Remote Foot Temperature Monitoring for Prevention of Amputation in a Large Integrated Health Care System.

OBJECTIVE: We evaluated the effectiveness of remote foot temperature monitoring (RTM) in the Veterans Affairs health care system. RESEARCH DESIGN AND METHODS: We conducted a retrospective cohort study that included 924 eligible patients enrolled in RTM between 2019 and 2021 who were matched up to 3:1 to 2,757 nonenrolled comparison patients. We used conditional Cox regression to estimate adjusted cause-specific hazard ratios (aHRs) and corresponding 95% CIs for lower-extremity amputation (LEA) as the primary outcome and all-cause hospitalization and death as secondary outcomes. RESULTS: RTM was not associated with LEA incidence (aHR 0.92, 95% CI 0.62-1.37) or all-cause hospitalization (aHR 0.97, 95% CI 0.82-1.14) but was inversely associated (reduced risk) with death (aHR 0.63, 95% CI 0.49-0.82). CONCLUSIONS: This study does not provide support that RTM reduces the risk of LEA or all-cause hospitalization in individuals with a history of diabetic foot ulcer. Randomized controlled trials can overcome important limitations.

The Development and Usability of the AMPREDICT Decision Support Tool: A Mixed Methods Study.

OBJECTIVE: Amputation level decision making in patients with chronic limb threatening ischaemia is challenging. Currently, evidence relies on published average population risks rather than individual patient risks. The result is significant variation in the distribution of amputation levels across health systems, geographical regions, and time. Clinical decision support has been shown to enhance decision making, especially complex decision making. The goal of this study was to translate the previously validated AMPREDICT prediction models by developing and testing the usability of the AMPREDICT Decision Support Tool (DST), a novel, web based, clinical DST that calculates individual one year post-operative risk of death, re-amputation, and probability of achieving independent mobility by amputation level. METHODS: A mixed methods approach was used. Previously validated prediction models were translated into a web based DST with additional content and format developed by an expert panel. Tool usability was assessed using the Post-Study System Usability Questionnaire (PSSUQ; a 16 item scale with scores ranging from 1 to 7, where lower scores indicate greater usability) by 10 clinician end users from diverse specialties, sex, geography, and clinical experience. Think aloud, semi-structured, qualitative interviews evaluated the AMPREDICT DST's look and feel, user friendliness, readability, functionality, and potential implementation challenges. RESULTS: The PSSUQ overall and subscale scores were favourable, with a mean overall total score of 1.57 (standard deviation [SD] 0.69) and a range from 1.00 to 3.21. The potential clinical utility of the DST included (1) assistance in counselling patients on amputation level decisions, (2) setting outcome expectations, and (3) use as a tool in the academic environment to facilitate understanding of factors that contribute to various outcome risks. CONCLUSION: After extensive iterative development and testing, the AMPREDICT DST was found to demonstrate strong usability characteristics and clinical relevance. Further evaluation will benefit from integration into an electronic health record with assessment of its impact on physician and patient shared amputation level decision making.

Cardiac myosin binding protein-C phosphorylation accelerates ß-cardiac myosin detachment rate in mouse myocardium.

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that influences sarcomere stiffness and modulates cardiac contraction-relaxation through its phosphorylation. Phosphorylation of cMyBP-C and ablation of cMyBP-C have been shown to increase the rate of MgADP release in the acto-myosin cross-bridge cycle in the intact sarcomere. The influence of cMyBP-C on Pi-dependent myosin kinetics has not yet been examined. We investigated the effect of cMyBP-C, and its phosphorylation, on myosin kinetics in demembranated papillary muscle strips bearing the ß-cardiac myosin isoform from nontransgenic and homozygous transgenic mice lacking cMyBP-C. We used quick stretch and stochastic length-perturbation analysis to characterize rates of myosin detachment and force development over 0-12 mM Pi and at maximal (pCa 4.8) and near-half maximal (pCa 5.75) Ca2+ activation. Protein kinase A (PKA) treatment was applied to half the strips to probe the effect of cMyBP-C phosphorylation on Pi sensitivity of myosin kinetics. Increasing Pi increased myosin cross-bridge detachment rate similarly for muscles with and without cMyBP-C, although these rates were higher in muscle without cMyBP-C. Treating myocardial strips with PKA accelerated detachment rate when cMyBP-C was present over all Pi, but not when cMyBP-C was absent. The rate of force development increased with Pi in all muscles. However, Pi sensitivity of the rate force development was reduced when cMyBP-C was present versus absent, suggesting that cMyBP-C inhibits Pi-dependent reversal of the power stroke or stabilizes cross-bridge attachment to enhance the probability of completing the power stroke. These results support a functional role for cMyBP-C in slowing myosin detachment rate, possibly through a direct interaction with myosin or by altering strain-dependent myosin detachment via cMyBP-C-dependent stiffness of the thick filament and myofilament lattice. PKA treatment reduces the role for cMyBP-C to slow myosin detachment and thus effectively accelerates ß-myosin detachment in the intact myofilament lattice.NEW & NOTEWORTHY Length perturbation analysis was used to demonstrate that ß-cardiac myosin characteristic rates of detachment and recruitment in the intact myofilament lattice are accelerated by Pi, phosphorylation of cMyBP-C, and the absence of cMyBP-C. The results suggest that cMyBP-C normally slows myosin detachment, including Pi-dependent detachment, and that this inhibition is released with phosphorylation or absence of cMyBP-C.

How patients interpret early signs of foot problems and reasons for delays in care: Findings from interviews with patients who have undergone toe amputations.

AIMS: To describe how patients respond to early signs of foot problems and the factors that result in delays in care. METHODS: Semi-structured interviews were conducted with a large sample of Veterans from across the United States with diabetes mellitus who had undergone a toe amputation. Data were analyzed using inductive content analysis. RESULTS: We interviewed 61 male patients. Mean age was 66 years, 41% were married, and 37% had a high school education or less. The patient-level factors related to delayed care included: 1) not knowing something was wrong, 2) misinterpreting symptoms, 3) "sudden" and "unexpected" illness progression, and 4) competing priorities getting in the way of care-seeking. The system-level factors included: 5) asking patients to watch it, 6) difficulty getting the right type of care when needed, and 7) distance to care and other transportation barriers. CONCLUSION: A confluence of patient factors (e.g., not examining their feet regularly or thoroughly and/or not acting quickly when they noticed something was wrong) and system factors (e.g., absence of a mechanism to support patient's appraisal of symptoms, lack of access to timely and convenient-located appointments) delayed care. Identifying patient- and system-level interventions that can shorten or eliminate care delays could help reduce rates of limb loss.

The N terminus of myosin-binding protein C extends toward actin filaments in intact cardiac muscle.

Myosin and actin filaments are highly organized within muscle sarcomeres. Myosin-binding protein C (MyBP-C) is a flexible, rod-like protein located within the C-zone of the sarcomere. The C-terminal domain of MyBP-C is tethered to the myosin filament backbone, and the N-terminal domains are postulated to interact with actin and/or the myosin head to modulate filament sliding. To define where the N-terminal domains of MyBP-C are localized in the sarcomere of active and relaxed mouse myocardium, the relative positions of the N terminus of MyBP-C and actin were imaged in fixed muscle samples using super-resolution fluorescence microscopy. The resolution of the imaging was enhanced by particle averaging. The images demonstrate that the position of the N terminus of MyBP-C is biased toward the actin filaments in both active and relaxed muscle preparations. Comparison of the experimental images with images generated in silico, accounting for known binding partner interactions, suggests that the N-terminal domains of MyBP-C may bind to actin and possibly the myosin head but only when the myosin head is in the proximity of an actin filament. These physiologically relevant images help define the molecular mechanism by which the N-terminal domains of MyBP-C may search for, and capture, molecular binding partners to tune cardiac contractility.

A high-throughput screening identifies ZNF418 as a novel regulator of the ubiquitin-proteasome system and autophagy-lysosomal pathway.

The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are two major protein degradation pathways in eukaryotic cells. Initially considered as two independent pathways, there is emerging evidence that they can work in concert. As alterations of UPS and ALP function can contribute to neurodegenerative disorders, cancer and cardiac disease, there is great interest in finding targets that modulate these catabolic processes. We undertook an unbiased, total genome high-throughput screen to identify novel effectors that regulate both the UPS and ALP. We generated a stable HEK293 cell line expressing a UPS reporter (UbG76V-mCherry) and an ALP reporter (GFP-LC3) and screened for genes for which knockdown increased both UbG76V-mCherry intensity and GFP-LC3 puncta. With stringent selection, we isolated 80 candidates, including the transcription factor ZNF418 (ZFP418 in rodents). After screen validation with Zfp418 overexpression in HEK293 cells, we evaluated Zfp418 knockdown and overexpression in neonatal rat ventricular myocytes (NRVMs). Endogenous and overexpressed ZFP418 were localized in the nucleus. Subsequent experiments showed that ZFP418 negatively regulates UPS and positively regulates ALP activity in NRVMs. RNA-seq from Zfp418 knockdown revealed altered gene expression of numerous ubiquitinating and deubiquitinating enzymes, decreased expression of autophagy activators and initiators and increased expression of autophagy inhibitors. We found that ZPF418 activated the promoters of Dapk2 and Fyco1, which are involved in autophagy. RNA-seq from Zfp418 knockdown revealed accumulation of several genes involved in cardiac development and/or hypertrophy. In conclusion, our study provides evidence that ZNF418 activates the ALP, inhibits the UPS and regulates genes associated with cardiomyocyte structure/function.Abbreviations: ACTN2, actinin alpha 2; ALP, autophagy-lysosomal pathway; COPB1, COPI coat complex subunit beta 1; DAPK2, death associated protein kinase 2; FYCO1, FYVE and coiled-coil domain autophagy adaptor 1; HEK293, human embryonic kidney cells 293; HTS, high-throughput screen; LC3, microtubule associated protein 1 light chain 3; NRVMs, neonatal rat ventricular myocytes; RNA-seq, RNA sequencing; RPS6, ribosomal protein S6; TNNI3, troponin I, cardiac 3; UPS, ubiquitin-proteasome system; shRNA, short hairpin RNA; SQSTM1/p62, sequestosome 1; VPS28, VPS28 subunit of ESCRT-I; ZNF418/ZFP418, zinc finger protein 418.

CYLD exaggerates pressure overload-induced cardiomyopathy via suppressing autolysosome efflux in cardiomyocytes.

Deubiquitinating enzymes (DUBs) appear to be a new class of regulators of cardiac homeostasis and disease. However, DUB-mediated signaling in the heart is not well understood. Herein we report a novel mechanism by which cylindromatosis (CYLD), a DUB mediates cardiac pathological remodeling and dysfunction. Cardiomyocyte-restricted (CR) overexpression of CYLD (CR-CYLD) did not cause gross health issues and hardly affected cardiac function up to age of one year in both female and male mice at physiological conditions. However, CR-CYLD overexpression exacerbated pressure overload (PO)-induced cardiac dysfunction associated with suppressed cardiac hypertrophy and increased myocardial apoptosis in mice independent of the gender. At the molecular level, CR-CYLD overexpression enhanced PO-induced increases in poly-ubiquitinated proteins marked by lysine (K)48-linked ubiquitin chains and autophagic vacuoles containing undegraded contents while suppressing autophagic flux. Augmentation of cardiac autophagy via CR-ATG7 overexpression protected against PO-induced cardiac pathological remodeling and dysfunction in both female and male mice. Intriguingly, CR-CYLD overexpression switched the CR-ATG7 overexpression-dependent cardiac protection into myocardial damage and dysfunction associated with increased accumulation of autophagic vacuoles containing undegraded contents in the heart. Genetic manipulation of Cyld in combination with pharmacological modulation of autophagic functional status revealed that CYLD suppressed autolysosomal degradation and promoted cell death in cardiomyocytes. In addition, Cyld gene gain- and/or loss-of-function approaches in vitro and in vivo demonstrated that CYLD mediated cardiomyocyte death associated with impaired reactivation of mechanistic target of rapamycin complex 1 (mTORC1) and upregulated Ras genes from rat brain 7 (Rab7), two key components for autolysosomal degradation. These results demonstrate that CYLD serves as a novel mediator of cardiac pathological remodeling and dysfunction by suppressing autolysosome efflux in cardiomyocytes. Mechanistically, it is most likely that CYLD suppresses autolysosome efflux via impairing mTORC1 reactivation and interrupting Rab7 release from autolysosomes in cardiomyocytes.

Risk of Ipsilateral Reamputation Following an Incident Toe Amputation Among U.S. Military Veterans With Diabetes, 2005-2016.

OBJECTIVE: To assess whether the risk of subsequent lower-limb amputations and death following an initial toe amputation among individuals with diabetes has changed over time and varies by demographic characteristics and geographic region. RESEARCH DESIGN AND METHODS: Using Veterans Health Administration (VHA) electronic medical records from 1 October 2004 to 30 September 2016, we determined risk of subsequent ipsilateral minor and major amputation within 1 year after an initial toe/ray amputation among veterans with diabetes. To assess changes in the annual rate of subsequent amputation over time, we estimated age-adjusted incidence of minor and major subsequent ipsilateral amputation for each year, separately for African Americans (AAs) and whites. Geographic variation was assessed across VHA markets (n = 89) using log-linear Poisson regression models adjusting for age and ethnoracial category. RESULTS: Among 17,786 individuals who had an initial toe amputation, 34% had another amputation on the same limb within 1 year, including 10% who had a major ipsilateral amputation. Median time to subsequent ipsilateral amputation (minor or major) was 36 days. One-year risk of subsequent major amputation decreased over time, but risk of subsequent minor amputation did not. Risk of subsequent major ipsilateral amputation was higher in AAs than whites. After adjusting for age and ethnoracial category, 1-year risk of major subsequent amputation varied fivefold across VHA markets. CONCLUSIONS: Nearly one-third of individuals require reamputation following an initial toe amputation, although risks of subsequent major ipsilateral amputation have decreased over time. Nevertheless, risks remain particularly high for AAs and vary substantially geographically.

Ube2v1 Positively Regulates Protein Aggregation by Modulating Ubiquitin Proteasome System Performance Partially Through K63 Ubiquitination.

RATIONALE: Compromised protein quality control can result in proteotoxic intracellular protein aggregates in the heart, leading to cardiac disease and heart failure. Defining the participants and understanding the underlying mechanisms of cardiac protein aggregation is critical for seeking therapeutic targets. We identified Ube2v1 (ubiquitin-conjugating enzyme E2 variant 1) in a genome-wide screen designed to identify novel effectors of the aggregation process. However, its role in the cardiomyocyte is undefined. OBJECTIVE: To assess whether Ube2v1 regulates the protein aggregation caused by cardiomyocyte expression of a mutant αB crystallin (CryABR120G) and identify how Ube2v1 exerts its effect. METHODS AND RESULTS: Neonatal rat ventricular cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB (CryABWT) or CryABR120G. Subsequently, loss- and gain-of-function experiments were performed. Ube2v1 knockdown decreased aggregate accumulation caused by CryABR120G expression. Overexpressing Ube2v1 promoted aggregate formation in CryABWT and CryABR120G-expressing neonatal rat ventricular cardiomyocytes. Ubiquitin proteasome system performance was analyzed using a ubiquitin proteasome system reporter protein. Ube2v1 knockdown improved ubiquitin proteasome system performance and promoted the degradation of insoluble ubiquitinated proteins in CryABR120G cardiomyocytes but did not alter autophagic flux. Lys (K) 63-linked ubiquitination modulated by Ube2v1 expression enhanced protein aggregation and contributed to Ube2v1's function in regulating protein aggregate formation. Knocking out Ube2v1 exclusively in cardiomyocytes by using AAV9 (adeno-associated virus 9) to deliver multiplexed single guide RNAs against Ube2v1 in cardiac-specific Cas9 mice alleviated CryABR120G-induced protein aggregation, improved cardiac function, and prolonged lifespan in vivo. CONCLUSIONS: Ube2v1 plays an important role in protein aggregate formation, partially by enhancing K63 ubiquitination during a proteotoxic stimulus. Inhibition of Ube2v1 decreases CryABR120G-induced aggregate formation through enhanced ubiquitin proteasome system performance rather than autophagy and may provide a novel therapeutic target to treat cardiac proteinopathies.

Calpain-2 promotes MKP-1 expression protecting cardiomyocytes in both in vitro and in vivo mouse models of doxorubicin-induced cardiotoxicity.

We recently reported that doxorubicin decreased the expression of calpain-1/2, while inhibition of calpain activity promoted doxorubicin-induced cardiac injury in mice. In this study, we investigated whether and how elevation of calpain-2 could affect doxorubicin-triggered cardiac injury. Transgenic mice with inducible cardiomyocyte-specific expression of calpain-2 were generated. An acute cardiotoxicity was induced in both transgenic mice and their relevant wild-type littermates by injection of a single dose of doxorubicin (20 mg/kg) and cardiac injury was analyzed 5 days after doxorubicin injection. Cardiomyocyte-specific up-regulation of calpain-2 did not induce any adverse cardiac phenotypes under physiological conditions by age 3 months, but significantly reduced myocardial injury and improved myocardial function in doxorubicin-treated mice. Cardiac protection of calpain-2 up-regulation was also observed in a mouse model of chronic doxorubicin cardiotoxicity. Up-regulation of calpain-2 increased the protein levels of mitogen activated protein kinase phosphatase-1 (MKP-1) in cultured mouse cardiomyocytes and heart tissues. Over-expression of MKP-1 prevented, whereas knockdown of MKP-1 augmented doxorubicin-induced apoptosis in cultured cardiomyocytes. Moreover, knockdown of MKP-1 offset calpain-2-elicited protective effects against doxorubicin-induced injury in cultured cardiomyocytes. Mechanistically, up-regulation of calpain-2 reduced the protein levels of phosphatase and tensin homolog and consequently promoted Akt activation, leading to increased MKP-1 protein steady-state levels by inhibiting its degradation. Collectively, this study reveals a new role of calpain-2 in promoting MKP-1 expression via phosphatase and tensin homolog/Akt signaling. This study also suggests that calpain-2/MKP-1 signaling may represent new therapeutic targets for doxorubicin-induced cardiac injury.

In Vivo Remodeling of an Extracellular Matrix Cardiac Patch in an Ovine Model.

Lack of an ideal patch material for cardiac repairs continues to challenge congenital heart surgeons. The current materials are unable to grow and result in scarring, contraction, and arrhythmias. An acellular extracellular matrix (ECM) patch derived from porcine small intestinal submucosa has demonstrated remodeling potential when used to repair various tissues. This study investigated the in vivo electrophysiologic, mechanical, and histological properties of an ECM patch used to repair a right-ventricular (RV) wall defect in a growing ovine model. A full-thickness, 2 × 2 cm RV defect was created in 11 juvenile sheep and repaired with an ECM patch. Longitudinal RV three-dimensional-electrical mapping, magnetic resonance imaging (MRI), and histological analysis were performed at 3, 6, 9, and 12 months. Three-dimensional mapping demonstrated consistent conduction across the patch with little to no difference in voltage, but conduction velocity was consistently less than native myocardium. Magnetic resonance imaging revealed changing strain properties of the patch which by 9-12 months resembled native tissue. Histologic analysis at 3 months demonstrates cardiomyocyte degeneration and partial replacement via proliferation of connective tissue cells that were predominately fibroblasts and smooth muscle cells. There was marked neovascularization and an absence of calcification at 12 months. Over time, the ECM patch remained viable with stable muscle at the edges. In growing sheep, an ECM patch becomes a viable tissue and remains so up to at least a year. Although ECM demonstrates some functional aspects of remodeling to native myocardium, histologically it remained immature.

Myofibroblast-Specific TGFß Receptor II Signaling in the Fibrotic Response to Cardiac Myosin Binding Protein C-Induced Cardiomyopathy.

RATIONALE: Hypertrophic cardiomyopathy occurs with a frequency of about 1 in 500 people. Approximately 30% of those affected carry mutations within the gene encoding cMyBP-C (cardiac myosin binding protein C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C (Mybpc340kDa). Expression of the 40kDa fragment in mouse cardiomyocytes leads to hypertrophy, fibrosis, and heart failure. Here we use genetic approaches to establish a causal role for excessive myofibroblast activation in a slow, progressive genetic cardiomyopathy-one that is driven by a cardiomyocyte-intrinsic genetic perturbation that models an important human disease. OBJECTIVE: TGFß (transforming growth factor-ß) signaling is implicated in a variety of fibrotic processes, and the goal of this study was to define the role of myofibroblast TGFß signaling during chronic Mybpc340kDa expression. METHODS AND RESULTS: To specifically block TGFß signaling only in the activated myofibroblasts in Mybpc340kDa transgenic mice and quadruple compound mutant mice were generated, in which the TGFß receptor II (TßRII) alleles ( Tgfbr2) were ablated using the periostin ( Postn) allele, myofibroblast-specific, tamoxifen-inducible Cre ( Postnmcm) gene-targeted line. Tgfbr2 was ablated either early or late during pathological fibrosis. Early myofibroblast-specific Tgfbr2 ablation during the fibrotic response reduced cardiac fibrosis, alleviated cardiac hypertrophy, preserved cardiac function, and increased lifespan of the Mybpc340kDa transgenic mice. Tgfbr2 ablation late in the pathological process reduced cardiac fibrosis, preserved cardiac function, and prolonged Mybpc340kDa mouse survival but failed to reverse cardiac hypertrophy. CONCLUSIONS: Fibrosis and cardiac dysfunction induced by cardiomyocyte-specific expression of Mybpc340kDa were significantly decreased by Tgfbr2 ablation in the myofibroblast. Surprisingly, preexisting fibrosis was partially reversed if the gene was ablated subsequent to fibrotic deposition, suggesting that continued TGFß signaling through the myofibroblasts was needed to maintain the heart fibrotic response to a chronic, disease-causing cardiomyocyte-only stimulus.

Hypertrophic cardiomyopathy R403Q mutation in rabbit ß-myosin reduces contractile function at the molecular and myofibrillar levels.

In 1990, the Seidmans showed that a single point mutation, R403Q, in the human ß-myosin heavy chain (MHC) of heart muscle caused a particularly malignant form of familial hypertrophic cardiomyopathy (HCM) [Geisterfer-Lowrance AA, et al. (1990) Cell 62:999-1006.]. Since then, more than 300 mutations in the ß-MHC have been reported, and yet there remains a poor understanding of how a single missense mutation in the MYH7 gene can lead to heart disease. Previous studies with a transgenic mouse model showed that the myosin phenotype depended on whether the mutation was in an α- or ß-MHC backbone. This led to the generation of a transgenic rabbit model with the R403Q mutation in a ß-MHC backbone. We find that the in vitro motility of heterodimeric R403Q myosin is markedly reduced, whereas the actin-activated ATPase activity of R403Q subfragment-1 is about the same as myosin from a nontransgenic littermate. Single myofibrils isolated from the ventricles of R403Q transgenic rabbits and analyzed by atomic force microscopy showed reduced rates of force development and relaxation, and achieved a significantly lower steady-state level of isometric force compared with nontransgenic myofibrils. Myofibrils isolated from the soleus gave similar results. The force-velocity relationship determined for R403Q ventricular myofibrils showed a decrease in the velocity of shortening under load, resulting in a diminished power output. We conclude that independent of whether experiments are performed with isolated molecules or with ordered molecules in the native thick filament of a myofibril, there is a loss-of-function induced by the R403Q mutation in ß-cardiac myosin.

Cardiac Fibrosis in Proteotoxic Cardiac Disease is Dependent Upon Myofibroblast TGF -ß Signaling.

Background Transforming growth factor beta ( TGF -ß) is an important cytokine in mediating the cardiac fibrosis that often accompanies pathogenic cardiac remodeling. Cardiomyocyte-specific expression of a mutant αB-crystallin (Cry ABR120G), which causes human desmin-related cardiomyopathy, results in significant cardiac fibrosis. During onset of fibrosis, fibroblasts are activated to the so-called myofibroblast state and TGF -ß binding mediates an essential signaling pathway underlying this process. Here, we test the hypothesis that fibroblast-based TGF -ß signaling can result in significant cardiac fibrosis in a disease model of cardiac proteotoxicity that has an exclusive cardiomyocyte-based etiology. Methods and Results Against the background of cardiomyocyte-restricted expression of Cry ABR120G, we have partially ablated TGF -ß signaling in cardiac myofibroblasts to observe whether cardiac fibrosis is reduced despite the ongoing pathogenic stimulus of Cry ABR120G production. Transgenic Cry ABR120G mice were crossed with mice containing a floxed allele of TGF -ß receptor 2 ( Tgfbr2 f/f). The double transgenic animals were subsequently crossed to another transgenic line in which Cre expression was driven from the periostin locus ( Postn) so that Tgfbr2 would be ablated with myofibroblast conversion. Structural and functional assays were then used to determine whether general fibrosis was affected and cardiac function rescued in Cry ABR120G mice lacking Tgfbr2 in the myofibroblasts. Ablation of myofibroblast specific TGF -ß signaling led to decreased morbidity in a proteotoxic disease resulting from cardiomyocyte autonomous expression of Cry ABR120G. Cardiac fibrosis was decreased and hypertrophy was also significantly attenuated, with a significant improvement in survival probability over time, even though the primary proteotoxic insult continued. Conclusions Myofibroblast-targeted knockdown of Tgfbr2 signaling resulted in reduced fibrosis and improved cardiac function, leading to improved probability of survival.

Cardiac Dysfunction in the Sigma 1 Receptor Knockout Mouse Associated With Impaired Mitochondrial Dynamics and Bioenergetics.

Background The Sigma 1 receptor (Sigmar1) functions as an interorganelle signaling molecule and elicits cytoprotective functions. The presence of Sigmar1 in the heart was first reported on the basis of a ligand-binding assay, and all studies to date have been limited to pharmacological approaches using less-selective ligands for Sigmar1. However, the physiological function of cardiac Sigmar1 remains unknown. We investigated the physiological function of Sigmar1 in regulating cardiac hemodynamics using the Sigmar1 knockout mouse (Sigmar1-/-). Methods and Results Sigmar1-/- hearts at 3 to 4 months of age showed significantly increased contractility as assessed by left ventricular catheterization with stimulation by increasing doses of a ß1-adrenoceptor agonist. Noninvasive echocardiographic measurements were also used to measure cardiac function over time, and the data showed the development of cardiac contractile dysfunction in Sigmar1 -/- hearts as the animals aged. Histochemistry demonstrated significant cardiac fibrosis, collagen deposition, and increased periostin in the Sigmar1 -/- hearts compared with wild-type hearts. Ultrastructural analysis of Sigmar1-/- cardiomyocytes revealed an irregularly shaped, highly fused mitochondrial network with abnormal cristae. Mitochondrial size was larger in Sigmar1-/- hearts, resulting in decreased numbers of mitochondria per microscopic field. In addition, Sigmar1-/- hearts showed altered expression of mitochondrial dynamics regulatory proteins. Real-time oxygen consumption rates in isolated mitochondria showed reduced respiratory function in Sigmar1-/- hearts compared with wild-type hearts. Conclusions We demonstrate a potential function of Sigmar1 in regulating normal mitochondrial organization and size in the heart. Sigmar1 loss of function led to mitochondrial dysfunction, abnormal mitochondrial architecture, and adverse cardiac remodeling, culminating in cardiac contractile dysfunction.

Aberrant Mitochondrial Fission Is Maladaptive in Desmin Mutation-Induced Cardiac Proteotoxicity.

BACKGROUND: Desmin filament proteins interlink the contractile myofibrillar apparatus with mitochondria, nuclei and the sarcolemma. Mutations in the human desmin gene cause cardiac disease, remodeling, and heart failure but the pathophysiological mechanisms remain unknown. METHODS AND RESULTS: Cardiomyocyte-specific overexpression of mutated desmin (a 7 amino acid deletion R172-E178, D7-Des Tg) causes accumulations of electron-dense aggregates and myofibrillar degeneration associated with cardiac dysfunction. Though extensive studies demonstrated that these altered ultrastructural changes cause impairment of cardiac contractility, the molecular mechanism of cardiomyocyte death remains elusive. In the present study, we report that the D7-Des Tg mouse hearts undergo aberrant mitochondrial fission associated with increased expression of mitochondrial fission regulatory proteins. Mitochondria isolated from D7-Des Tg hearts showed decreased mitochondrial respiration and increased apoptotic cell death. Overexpression of mutant desmin by adenoviral infection in cultured cardiomyocytes led to increased mitochondrial fission, inhibition of mitochondrial respiration, and activation of cellular toxicity. Inhibition of mitochondrial fission by mitochondrial division inhibitor mdivi-1 significantly improved mitochondrial respiration and inhibited cellular toxicity associated with D7-Des overexpression in cardiomyocytes. CONCLUSIONS: Aberrant mitochondrial fission results in mitochondrial respiratory defects and apoptotic cell death in D7-Des Tg hearts. Inhibition of aberrant mitochondrial fission using mitochondrial division inhibitor significantly preserved mitochondrial function and decreased apoptotic cell death. Taken together, our study shows that maladaptive aberrant mitochondrial fission causes desminopathy-associated cellular dysfunction.