Contractile Deficit Research Articles

Cardiovascular and arrhythmic manifestations of Bartter's and Gitelman's syndromes: do not forget the heart. A narrative literature review.

Bartter's and Gitelman's syndromes (BS/GS) are genetically determined kidney tubulopathies leading to electrolyte and neurohormonal abnormalities. Although considered benign entities, major adverse cardiovascular events may complicate both syndromes, in form of ventricular arrhythmias leading to palpitations, syncope or sudden cardiac death, microvascular cardiac dysfunction and exercise-induced myocardial contractile deficit. The mechanisms leading to cardiovascular complications are not only driven by chronic electrolyte abnormalities, i.e. chronic hypokalemia and hypomagnesemia, but also by neurohormonal alterations that can impair vascular tone and myocardial contractility. In presence of triggering factors, BS/GS patients may experience a spectrum of cardiac arrhythmias necessitating prompt diagnosis and treatment. The aim of this review is to explore the pathophysiological mechanisms of BS and GS, highlighting those responsible for cardiovascular involvement, and to analyze the spectrum of associated cardiovascular complications. This highlights the importance of an integrated shared management of GS/BS patients between Nephrologist and Cardiologist.

Abstract Mo092: Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as an In Vitro Model to Evaluate the Efficacy of a Gene Replacement Therapy for Plakophilin-2- Associated Arrhythmogenic Right Ventricular Cardiomyopathy

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disorder characterized by severe arrhythmias and heart dysfunction. Mutations in desmosome gene Plakophilin-2 ( PKP2 ) account for 40% of ARVC cases, with current palliative therapies failing to address the genetic cause. Herein, we generated an in vitro ARVC model using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) harboring a pathogenic PKP2 variant (c.2146G>C), as a platform to test a PKP2 -gene replacement approach developed at Tenaya Therapeutics for the treatment of PKP2 -associated ARVC patients. Methods: Three isogenic human iPSC lines (wild-type, heterozygous and homozygous PKP2 mutant) were differentiated to iPSC-CM; and monolayers and engineered heart tissues (EHTs) were generated. Gene and protein expression were assessed by RNA-Seq/RT-qPCR, and immunocytochemistry, respectively. Contractility, electrophysiology and calcium handling were measured. A proprietary adeno-associated virus gene therapy (AAV9: PKP2 ) was used to transduce human iPSC-CM, and changes in gene and protein expression, and contractile function were evaluated. Results: A human iPSC-CM beating monolayer was achieved for all three lines. PKP2 expression was depleted in a genotype-dependent manner, and desmosome structure was disrupted in the mutant lines. Functional assessment of human iPSC-CM monolayers showed impaired contractile properties and abnormal electrophysiological and calcium transients in the mutant lines, such as decreased contraction amplitude and prolonged field and action potential duration. Transcriptional analysis revealed changes in desmosome, gap junctions, sarcomere, ion channels, metabolic and apoptosis gene expression. Characterization of EHTs displayed a deficit in contractility, slower action and field potential kinetics and irregular calcium homeostasis in the mutant lines. The administration of AAV9: PKP2 to the mutant lines restored ion channel and desmosome gene and protein expression, and contractile function. Conclusions: Our PKP2 human iPSC-CM disease model recapitulated the main hallmarks of ARVC phenotype. Administration of AAV9: PKP2 restored desmosome protein expression and contractility. This model lays a foundation for the understanding of the underlying molecular pathophysiological mechanisms of PKP2 -ARVC, and the potential for AAV9: PKP2 as a one-time dose to correct the genetic cause of disease in individuals with PKP2 -associated ARVC.

Susceptibility to innate immune activation in genetically-mediated myocarditis.

Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment for myocarditis is often supportive. Mutations in DSP, a gene encoding the desmosomal protein desmoplakin, have been increasingly implicated in myocarditis with biomarkers and pathological features indistinguishable from other forms of myocarditis. DSP-associated myocarditis can progress to dilated cardiomyopathy with heightened arrhythmia risk. To model the cardiomyocyte aspects of DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues (EHTs) from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients and gene-edited healthy control hiPSC lines. Homozygous and heterozygous DSP disrupted EHTs were generated to contain 90% hiPSC-CMs and 10% healthy control human cardiac fibroblasts. We measured innate immune activation and function at baseline and in response to Toll-like receptor (TLR) stimulation in EHTs. At baseline, DSP-/- EHTs displayed a transcriptomic signature of immune activation which was mirrored by EHT cytokine release. Importantly, DSP-/- EHTs were hypersensitive to TLR stimulation demonstrating greater contractile function impairment compared to isogenic controls. Compared to homozygous DSP-/- EHTs, heterozygous DSP patient-derived EHTs had less functionally impairment but also displayed heightened sensitivity to TLR stimulation. When subjected to strain, heterozygous DSP EHTs developed greater functional deficit indicating reduced contractile reserve compared to healthy control. Colchicine or NFΚB inhibitors improved baseline force production and strain-induced force deficits in DSP EHTs. Genomic correction of DSP p.R1951X using adenine base editing reduced inflammatory biomarker release from EHTs. Genetic reduction of DSP renders cardiomyocytes susceptible to innate immune activation and strain-dependent contractile deficits. EHTs replicate electrical and contractile phenotypes seen in human myocarditis implicating cytokine release as a key part of the myogenic susceptibility to inflammation. This heightened innate immune activation and sensitivity is a target for clinical intervention.

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.

Cardiac tissue model of immune-induced dysfunction reveals the role of free mitochondrial DNA and the therapeutic effects of exosomes.

Despite tremendous progress in the development of mature heart-on-a-chip models, human cell-based models of myocardial inflammation are lacking. Here, we bioengineered a vascularized heart-on-a-chip with circulating immune cells to model severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced acute myocarditis. We observed hallmarks of coronavirus disease (COVID-19)-induced myocardial inflammation, as the presence of immune cells augmented the secretion of proinflammatory cytokines, triggered progressive impairment of contractile function, and altered intracellular calcium transients. An elevation of circulating cell-free mitochondrial DNA (ccf-mtDNA) was measured first in the heart-on-a-chip and then validated in COVID-19 patients with low left ventricular ejection fraction, demonstrating that mitochondrial damage is an important pathophysiological hallmark of inflammation-induced cardiac dysfunction. Leveraging this platform in the context of SARS-CoV-2-induced myocardial inflammation, we established that administration of endothelial cell-derived exosomes effectively rescued the contractile deficit, normalized calcium handling, elevated the contraction force, and reduced the ccf-mtDNA and cytokine release via Toll-like receptor-nuclear factor κB signaling axis.

Abstract EC.04: The Effects Of Cardiac Troponin Activator CK-358 On Cardiac Contractility In A Mouse Model Of Genetic Dilated Cardiomyopathy

Genetic dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. However, most treatments do not address the underlying contractile deficit caused by the disease. In an effort to create more targeted DCM therapeutics, novel small molecule drugs known as sarcomeric modulators have been of recent interest. In a recent study, a cardiac troponin activator was shown to improve cardiac muscle contractility without adversely affecting energetics or diastolic function. Here, we aim to elucidate the mechanism by which the cardiac troponin activator CK-3827358 (CK-358) is able to recover contractile function in a mouse model of DCM with a mutation at residue 61 of cardiac troponin C (cTnC I61Q). We measured steady state force and k tr , a measure of crossbridge cycling kinetics, in demembranated ventricular tissue from control and cTnC I61Q mice in solutions with increasing concentrations of calcium, ranging from pCa 9 to pCa 4. The cTnC I61Q mutation significantly decreased calcium sensitivity versus control (pCa 50 : 5.48 ± 0.06 vs 5.75 ± 0.01). Compared to controls, CK-358 (1 μM) significantly increased calcium sensitivity (pCa 50 ) in tissue from both cTnC I61Q and control mice (6.03 ± 0.06 and 6.50 ± 0.18, P=0.005) without any change in maximum force. CK-358 also significantly increased k tr , possibly suggesting activation of thin filaments. Next, we used a fluorescently labeled human cardiac troponin C (HcTnC) conjugated to N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) combined with subunits T and I to make a full HcTn complex. We performed calcium titrations with the HcTn-IANBD and saw no change in fluorescence in the control protein with and without CK-358. Finally, we performed HcTnC titrations with HcTnI and saw no change in fluorescence at maximum, sub-maximum, and minimum calcium concentrations. Overall, we demonstrate that cardiac troponin activator CK-358 did not alter calcium binding to HcTnC or its interaction with HcTnI. However, it restores the calcium sensitivity of cTnC I61Q DCM ventricular sarcomeres. Future studies will examine the effects of CK-358 on calcium binding in decorated thin filaments and measure activation using stopped flow spectroscopy in this model of DCM.

In this Issue

HomeCirculation ResearchVol. 132, No. 11In this Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn this Issue Originally published25 May 2023https://doi.org/10.1161/RES.0000000000000613Circulation Research. 2023;132:1425is related toEnhanced Mitochondria-SR Tethering Triggers Adaptive Cardiac Muscle RemodelingMechanism of Tumor-Platelet Communications in CancerTranscriptomic and Proteomic of Gastrocnemius Muscle in Peripheral Artery DiseaseTranscriptomic and Proteomic of Gastrocnemius Muscle in Peripheral Artery Disease (p 1428)Download figureDownload PowerPointFerrucci et al identify dysregulated pathways in the muscles of peripheral artery disease patients.Peripheral artery disease (PAD) occurs when atherosclerosis causes narrowing of the blood vessels supplying the lower extremities. Aside from leg pain during walking, the hypoxia causes damage to the muscles and there are no effective remedies. To look for potential drug targets, Ferrucci and colleagues performed transcriptomics and proteomics on biopsy samples from calf muscles of 31 people with PAD and 29 people without. They compared more than 20,000 transcripts and 6,000 proteins and from computational analyses found that PAD patient muscles exhibited ramped up inflammation, hypoxia responses, apoptosis, fibrosis and angiogenesis. They also unexpectedly found that while levels of mitochondrial proteins were increased in PAD muscles, levels of the related transcripts were decreased. The authors suggest this apparent discrepancy may be due to regulatory feedback wherein accumulation of dysfunctional mitochondria inhibits transcription of the related genes. Indeed, in PAD samples, the abundance of mitochondrial proteins did not correlate with mitochondrial activity while it did in controls. Whatever the reason, the results provide a wealth of data to inform drug development efforts aimed at preserving muscle health in PAD.Mechanism of Tumor-Platelet Communications in Cancer (p 1447)Download figureDownload PowerPointCancer-derived extracellular vesicles drive platelet activation, show Dudiki et al.Thrombosis is a major, sometimes fatal, complication of cancer. But how and why platelets—the key cells controlling clotting—become activated in cancer isn’t clear. Cancer patient platelets are known to accumulate tumor-derived factors and tumors are known to secrete procoagulant vesicles. Now, Dudiki and colleagues have tied these two findings together, showing that tumor-derived extracellular vesicles (EVs) are taken up by platelets, wherein they activate the cells. And they have also identified the EV key to the platelet door. The team applied EVs to cultured mouse platelets and found those derived from tumors were taken up more readily than synthetic or fibroblast-derived ones. The tumor EVs, but not the synthetic or fibroblast-derived ones were also able to induce platelet activation. The uptake of EVs led to accumulation of certain cancer-specific factors in the platelets and this was also true in vivo—when mice were injected with tumor EVs or had tumors implanted. The team went on to show that blocking CD63—a major EV surface protein—prevented EV-induced platelet activation in culture and thrombus formation in mice. They thus conclude that CD63 could be a clinical target for life-saving anti-thrombosis treatments in cancer patients.Enhanced Mitochondria-SR Tethering Triggers Adaptive Cardiac Muscle Remodeling (e171)Download figureDownload PowerPointEnhanced mitochondria-sarcoplasmic reticulum links improve heart resilience, say Nichtova et al.In cardiomyocytes, the sarcoplasmic reticulum (SR) is closely associated with mitochondria enabling calcium released by the former to enter the latter to power ATP production—known as excitation-bioenergetics coupling (EBC). Proteins tethers hold the organelles in proximity, and acute overexpression of such tethers improves EBC and corrects heart contractile deficits in mice with cardiomyopathy. The effects of long-term enhanced tethering are unknown, however, and might even be detrimental—causing excess reactive oxygen species from over-stimulated mitochondria. Now Nichtova and colleagues examine such a cardiomyocyte-specific scenario in mice, engineering a mitochondrial-SR tether that is expressed in the animals from birth. In addition to enhanced SR-mitochondrial connections, the heart cells of the animals exhibited increased contractility, strong calcium signaling and good EBC. Furthermore, when subjected to ischemia and reperfusion, the test animals’ hearts showed better recovery of contractility, smaller infarct size and less myocyte death than control hearts. They were also more resilient to adrenergic stress. The results suggest that long-term tethering is not detrimental and may have therapeutic potential for improving heart recovery after injury. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesEnhanced Mitochondria-SR Tethering Triggers Adaptive Cardiac Muscle RemodelingZuzana Nichtová, et al. Circulation Research. 2023;132:e171-e187Mechanism of Tumor-Platelet Communications in CancerTejasvi Dudiki, et al. Circulation Research. 2023;132:1447-1461Transcriptomic and Proteomic of Gastrocnemius Muscle in Peripheral Artery DiseaseLuigi Ferrucci, et al. Circulation Research. 2023;132:1428-1443 May 26, 2023Vol 132, Issue 11 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000613 Originally publishedMay 25, 2023 PDF download Advertisement

Right Ventricular Sarcomere Contractile Depression and the Role of Thick Filament Activation in Human Heart Failure With Pulmonary Hypertension.

Right ventricular (RV) contractile dysfunction commonly occurs and worsens outcomes in patients with heart failure with reduced ejection fraction and pulmonary hypertension (HFrEF-PH). However, such dysfunction often goes undetected by standard clinical RV indices, raising concerns that they may not reflect aspects of underlying myocyte dysfunction. We thus sought to characterize RV myocyte contractile depression in HFrEF-PH, identify those components reflected by clinical RV indices, and uncover underlying biophysical mechanisms. Resting, calcium-, and load-dependent mechanics were prospectively studied in permeabilized RV cardiomyocytes isolated from explanted hearts from 23 patients with HFrEF-PH undergoing cardiac transplantation and 9 organ donor controls. Unsupervised machine learning using myocyte mechanical data with the highest variance yielded 2 HFrEF-PH subgroups that in turn mapped to patients with decompensated or compensated clinical RV function. This correspondence was driven by reduced calcium-activated isometric tension in decompensated clinical RV function, whereas surprisingly, many other major myocyte contractile measures including peak power and myocyte active stiffness were similarly depressed in both groups. Similar results were obtained when subgroups were first defined by clinical indices, and then myocyte mechanical properties in each group compared. To test the role of thick filament defects, myofibrillar structure was assessed by x-ray diffraction of muscle fibers. This revealed more myosin heads associated with the thick filament backbone in decompensated clinical RV function, but not compensated clinical RV function, as compared with controls. This corresponded to reduced myosin ATP turnover in decompensated clinical RV function myocytes, indicating less myosin in a crossbridge-ready disordered-relaxed (DRX) state. Altering DRX proportion (%DRX) affected peak calcium-activated tension in the patient groups differently, depending on their basal %DRX, highlighting potential roles for precision-guided therapeutics. Last, increasing myocyte preload (sarcomere length) increased %DRX 1.5-fold in controls but only 1.2-fold in both HFrEF-PH groups, revealing a novel mechanism for reduced myocyte active stiffness and by extension Frank-Starling reserve in human heart failure. Although there are many RV myocyte contractile deficits in HFrEF-PH, commonly used clinical indices only detect reduced isometric calcium-stimulated force, which is related to deficits in basal and recruitable %DRX myosin. Our results support use of therapies to increase %DRX and enhance length-dependent recruitment of DRX myosin heads in such patients.

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

Iron supplementation improves skeletal muscle contractile properties in mice with chronic kidney disease.

Adult chronic kidney disease (CKD) patients commonly display decreased physical function, leading to increased mortality, which is exacerbated with iron deficiency and anemia. Interventions to mitigate CKD myopathy are scarce, potentially because the underlying contractile deficits have not been fully investigated. We examined single fiber force production, myosin-actin interactions and myofilament properties in soleus and extensor digitorum longus muscles of 16-week-old CKD mice without (CKD, N=12) and with iron supplementation (CKDFE, N=6, weekly injections of 0.2 mg/kg iron dextran) and controls (N=10). Late-stage CKD was induced by 0.2% adenine diet for 8 weeks. CKD with iron supplementation (CKDFE) improved anemia status compared to CKD, but not back to control levels. Maximally activated (pCa 4.5, 25°C) specific tension (maximal isometric force divided by cross-sectional area) was reduced 36-51% with CKD due, in a large part, to a reduction in strongly-bound myosin-actin cross-bridges in all myosin heavy chain (MHC) I, IIA, and IIB fibers. Iron supplementation improved specific tension and number of strongly-bound cross-bridges in MHC IIA and IIB fibers. Myosin-actin cross-bridge kinetics were slower with CKD in MHC I and IIA fibers, with iron supplementation restoring kinetics closer to control values in MHC IIA fibers. Higher hemoglobin levels were correlated to improvements in specific tension and strongly-bound cross-bridges in MHC IIA and IIB fibers, and with faster cross-bridge kinetics in MHC IIA fibers. However, CKD induced decreases in myofilament lattice stiffness for MHC IIA and IIB fibers were not improved with iron supplementation. Our findings show that iron supplementation improved various aspects of the cellular and molecular CKD-induced myopathy in fast-contracting fiber types. Thus, correction of iron imbalance and/or anemia may be an effective countermeasure to reduce contractile deficits and improve quality of life in late-stage CKD patients.

Pharmacological interventions improve myosin-actin cross-bridge function in mice with advanced chronic kidney disease.

Skeletal muscle fiber force production in adult mice with late-stage chronic kidney disease (CKD) is significantly impaired, although by different myofilament and myosin-actin cross-bridge disease responses in the various fiber types. To reverse some of the CKD-induced contractile deficits, we examined the effects of the ATP analog, 2-deoxyadenosine triphosphate (dATP), to enhance cross-bridge kinetics and the troponin sensitizer, Tirasemtiv (2 µM), to increase cross-bridge formation. Single fiber force production, myosin-actin interactions and myofilament properties were measured at 25°C in fibers from soleus and extensor digitorum longus muscles of 16-week-old CKD mice (N=5) and controls (N=5). Late-stage CKD was induced by 0.2% adenine diet for 8 weeks. dATP in myosin heavy chain (MHC) I and IIA fibers with CKD increased cross-bridge kinetics, returning them to control values or faster, leading to more strongly bound cross-bridges for maximal Ca2+-activation (pCa 4.5, pH 7.0, 5 mM Phosphate) and fatiguing (pCa 4.5, pH 6.2, 30 mM Phosphate) conditions. Specific tensions in CKD were also improved 12-21% under maximal and fatiguing conditions, except in maximally Ca2+-activated MHC I fibers, but fell short of control values. Notably, other studies have found that improvements of 13-25% in knee extensor strength in CKD patients improved their physical function, suggesting changes of this magnitude are clinically meaningful. At submaximal Ca2+-activation (pCa 6.0), Tirasemtiv in MHC IIB fibers with CKD increased myofilament stiffness 520%, number of strongly bound cross-bridges 705%, and specific tension 370%, returning specific tension back to control values. This study provides evidence that cellular and molecular contractile function in CKD may be enhanced through use of dATP or Tirasemtiv, which should be tested to potentially improve physical function and overall quality of life in late-stage CKD patients.

Skeletal muscle fiber force declines with chronic kidney disease in juvenile and adult mice.

Juvenile and adult chronic kidney disease (CKD) patients frequently have compromised physical performance, which worsens with disease severity. Examining the underlying mechanisms behind this contractile dysfunction can be difficult to impossible in humans, especially in juveniles, but can be accomplished using animal models. We characterized CKD-induced changes in cellular (single fiber force) and molecular (myosin-actin interactions, myofilament properties) function in soleus and extensor digitorum longus muscles of 6-week-old (juvenile, control = 5, CKD = 6) and 16-week-old (adults, control = 10, CKD = 12) mice. CKD was induced by 0.2% adenine diet for 3 weeks (early-stage CKD) for juveniles and 8 weeks (late-stage CKD) for adults. Maximally activated (pCa 4.5, 25°C) specific tension (maximal isometric force divided by cross-sectional area) was reduced in all myosin heavy chain (MHC) I, IIA, and IIB fibers in juvenile (23-30%) and adult (36-51%) mice with CKD due, in a large part, to a reduction in strongly-bound myosin-actin cross-bridges. Additionally, myofilament stiffness was reduced in MHC IIA fibers from juveniles and adults as well as MHC IIB fibers from adults, potentially reducing force transmission. For adults, myosin-actin interactions were slower in MHC I and IIA fibers. In juveniles, myosin-actin interactions were slower only in MHC IIA fibers but faster in MHC IIB fibers. Fiber cross-sectional area with CKD was increased or unchanged in all fiber types, except for a decrease in MHC IIB fibers from adults. Overall, juvenile early-stage and adult late-stage CKD showed similar molecular mechanisms leading to reduced force production and cross-bridge kinetics, although alterations were generally larger in adult late-stage CKD. Thus, physical function in juvenile early-stage and adult late-stage CKD is being impaired on the molecular level due to intrinsic contractile deficits independent of skeletal muscle atrophy.

Abstract 11471: Engineered Bacterial Sodium Channel Gene Therapy Prevents Contractile Deficit and Reduces Ventricular Arrhythmias in a Non-Human Primate Model of Myocardial Infarction

Introduction: Current therapies for myocardial infarction (MI) and sudden cardiac death are limited. The ability to enhance amplitudes of sodium current and calcium transient in cardiomyocytes could improve electrical synchrony and contractions of infarcted heart tissue to both prevent arrhythmias and improve pumping function. We hypothesized that adeno-associated virus (AAV) delivery of prokaryotic sodium channel (BacNa v ) can improve electrical and contractile function of infarcted hearts. Methods: MI in non-human primates (NHPs) was induced by 60-min ischemia / reperfusion injury. Animals were randomized to sham surgery (n=6) or MI followed by intramyocardial injection of AAV-BacNa v (n=6) or AAV-GFP (n=6) into the infarct area and border zone. Left ventricular (LV) function was assessed by echocardiogram at baseline, 1- and 4-week post-MI. Spontaneous arrhythmias were tracked by a subcutaneously implanted loop recorder during the 4-week study period. Results: At 4 weeks post-MI, BacNa v and GFP expression were detected in all treated animals (Fig. 1). Compared to sham animals, GFP-treated animals showed lower LV ejection fraction (LVEF, 37.4±3.4% vs 58.2±1.2%, p<0.0001) and increased LV end-systolic volume (3.8±0.57mL vs 2.1±0.25mL, p<0.05). BacNa v -treated animals showed significant increase in LVEF compared to the GFP group (48.3±3.4% vs 37.4±3.4%, p<0.05) and did not differ from the sham group. Compared to the GFP group, fewer BacNa v -treated animals displayed spontaneous arrhythmias (16.7% vs 100%, p<0.05) and had less arrhythmia episodes per animal (0.17±0.17 vs 2.17±0.6, p<0.01), while not differing in any measured electrophysiological parameters from the sham group. Conclusion: The AAV-mediated BacNa v expression in cardiomyocytes can alleviate cardiac contractile deficit and reduce ventricular arrhythmias in the setting of MI in an NHP model. BacNa v gene delivery may represent a promising therapeutic strategy for the treatment of MI.

Abstract 13724: Development of VS-041 as a Precision Medicine Targeting Cardiac-Relevant Matrix Metalloproteases (MMPs) in Heart Failure With Preserved Ejection Fraction (HFpEF)

Introduction. HFpEF is a heterogeneous syndrome, with several underlying etiologic and pathophysiologic factors. As “one size fits all” therapies are not equally efficacious across various HFpEF comorbidities, a precision medicine approach can offer a disease modifying treatment for HFpEF subpopulation stratified for specific molecular targets. Expression of MMP2, 9, and 13 in heart is elevated in HFpEF patients with hypertension, diabetes or coronary disease, and their levels in plasma correlate with survival, suggesting utility as predictive biomarkers. Genetic ablation of MMP2, 9, and 13 leads to a robust antifibrotic effect in the myocardium. Inhibition of MMP2, 9, and 13 directly regulates of Ca 2+ transients and protects sarcomere function. Hypothesis. We present preclinical profile of VS-041, a novel and narrow-spectrum MMP inhibitor, as a drug candidate for HFpEF patients with elevated levels of MMP2, 9, and 13. Methods and Results. VS-041, optimized with computer-aided drug design methods, shows single-digit IC 50 against MMP2 (1nM), MMP9 (2nM) and MMP13 (1nM), and maintains >300-fold selectivity against MMP1 and TACE. In isolated cardiomyocytes VS-041 reduces contractility deficit due to fast pacing. With high predicted metabolic bioavailability (MF%=90), >80% target occupancy by VS-041 is estimated after 50 mg/kg PO dose in rats. VS-041 demonstrated robust efficacy in Dahl Salt Sensitive rat model of hypertension and HFpEF after dosing at 25 or 50 mg/kg PO for 5 wks in a therapeutic mode (after 1-wk of high salt lead-in). VS-041 had no effect on blood pressure or ejection fraction confirming a direct effect on diastolic function. In Doppler mitral inflow, analysis of the isovolumic relaxation time and the E/A ratio of peak velocity of filling waves revealed 40% improvement in diastolic function. LV fibrosis was dose-proportionally decreased based on PSR morphometry. No adverse effects were observed after 5 weeks of dosing. Conclusions. Preclinical pharmacology and safety profile of VS-041 support its nomination as clinical candidate and initiation of IND-enabling studies. VS-041 has potential to be first-in-class precision medicine treatment for HFPEF patients with elevated levels of cardiac-relevant MMP2, 9 and 13.

Abstract P2120: Microdystrophin Design Comparisons In Dystrophin-Deficient Cardiomyocytes

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Dilated cardiomyopathy is the leading cause of death in DMD patients. Preclinical data show that smaller versions of dystrophin, called microdystrophins, can ameliorate disease progression. These transgenes, amenable to packaging into adeno-associated virus, have shown to be effective in improving skeletal muscle function; however, the functional benefit of microdystrophins in delaying heart failure is unknown. To identify the design principles that are important for addressing DMD heart dysfunction, three microdystrophin variants currently in clinical trials were tested in cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs) with deficiencies in dystrophin. Previously, we reported that mechanical contraction and calcium handling are impaired in DMD iPSC-CMs. To test the functional benefit of the microdystrophins, we performed traction force microscopy, that provides multiparametric measurements of contraction, and calcium imaging with a ratiometric calcium-binding dye. Our results show a partial rescue of contractile deficits and aberrant calcium handling, underscoring the need for new design variants that address cardiomyocyte dysfunction.

Abstract P2121: Modeling Genetic Dilated Cardiomyopathies With Engineered Heart Tissues From Patient-derived And Isogenic Mutant Induced Pluripotent Stem Cells

The availability of human induced pluripotent stem cells (hiPSCs) has offered the possibility to study human-derived models of different genetic strata of cardiomyopathy for mechanistic discovery and therapeutic development. However, the phenotypes derived from cardiomyocytes differentiated from hiPSCs in conventional 2-dimensional culture systems often fail to reproducibly model clinical presentations, thereby reducing the translatability of readouts from these assays. Here we report 3-dimensional engineered heart tissues (EHTs) produced with a variety of cell lines harboring patient-derived mutations (TTN, RBM20, BAG3) known to cause dilated cardiomyopathy (DCM) which recapitulate contractile deficits associated with DCM. Internal development of EHTs also recapitulate contractile defects with small interfering RNA (siRNA) knockdown models of haploinsufficiency across multiple disease states. These observed findings with EHTs suggests improved maturity in cardiomyocytes through the presentation of microenvironmental cues akin to those seen in vivo . With continued advances in EHT technologies and capabilities, this platform may serve to significantly reduce drug candidate attrition, as well as provide new insights into pathology, prototyping of therapeutic approaches, and mechanism of action that were previously difficult to obtain.

Iron Supplementation Improves Skeletal Muscle Contractile Properties in Mice with CKD.

Patients with chronic kidney disease (CKD) frequently have compromised physical performance, which increases their mortality; however, their skeletal muscle dysfunction has not been characterized at the single-fiber and molecular levels. Notably, interventions to mitigate CKD myopathy are scarce. The effect of CKD in the absence and presence of iron supplementation on the contractile function of individual skeletal muscle fibers from the soleus and extensor digitorum longus muscles was evaluated in 16-week-old mice. CKD was induced by the adenine diet, and iron supplementation was by weekly iron dextran injections. Maximally activated and fatigued fiber force production was decreased 24%-52% in untreated CKD, independent of size, by reducing strongly bound myosin/actin cross-bridges and/or decreasing myofilament stiffness in myosin heavy chain (MHC) I, IIA, and IIB fibers. Additionally, myosin/actin interactions in untreated CKD were slower for MHC I and IIA fibers and unchanged or faster in MHC IIB fibers. Iron supplementation improved anemia and did not change overall muscle mass in CKD mice. Iron supplementation ameliorated CKD-induced myopathy by increasing strongly bound cross-bridges, leading to improved specific tension, and/or returning the rate of myosin/actin interactions toward or equivalent to control values in MHC IIA and IIB fibers. Skeletal muscle force production was significantly reduced in untreated CKD, independent of fiber size, indicating that compromised physical function in patients is not solely due to muscle mass loss. Iron supplementation improved multiple aspects of CKD-induced myopathy, suggesting that timely correction of iron imbalance may aid in ameliorating contractile deficits in CKD patients.

Multifidus Muscle Contractility Deficit Was Not Specific to the Painful Side in Patients with Chronic Low Back Pain During Remission: A Cross-Sectional Study.

PurposeMorphology studies demonstrated that patients with chronic low back pain (CLBP) have bilateral multifidus muscle (LM) atrophy. This atrophy should result in LM contractility deficit bilaterally. Additionally, a recent study showed the effect of sex on LM thickness. Researchers proposed percentage LM contractility (LMCONT) as standardization to enable the comparison across participants. This study aimed to determine side-to-side difference in LMCONT and to determine the difference in LMCONT between males and females.Patients and MethodsTwenty-five healthy individuals (NoLBP group; 10 males and 15 females) and 35 with CLBP (CLBP group; 16 males and 19 females; 23 unilateral pain and 12 bilateral pain) were recruited. Ultrasound imaging was used to measure LM thickness at rest, during maximum voluntary isometric contraction, and during combined maximum voluntary isometric contraction with electrical stimulation. These data were used to calculate LMCONT. For unilateral CLBP, right and left LMCON were renamed to painful and non-painful sides.ResultsData demonstrated no significant difference (p > 0.05) between right (87.3 ± 13.7%) and left (87.2 ± 14.0%) in NoLBP, right (71.2 ± 15.7%) and left (76.5 ± 19.7%) in bilateral CLBP, and painful (70.3 ± 17.5%) and non-painful (77.7 ± 18.4%) in unilateral CLBP. No difference (p > 0.05) was found between males and females in both NoLBP (male 84.8 ± 6.5%, female 88.9 ± 15.4%) and CLBP groups (male 76.3 ± 15.5%, female 71.9 ± 14.0%).ConclusionThe findings suggested that LM contractility deficit in CLBP is not specific to painful side. No effect was found of sex on LM contractility. Therefore, we can use averaged LM activation across painful and non-painful sides and across males and females to compare between NoLBP and CLBP groups.

Cardiac myocyte intrinsic contractility and calcium handling deficits underlie heart organ dysfunction in murine cancer cachexia

Cachexia is a muscle wasting syndrome occurring in many advanced cancer patients. Cachexia significantly increases cancer morbidity and mortality. Cardiac atrophy and contractility deficits have been observed in patients and in animal models with cancer cachexia, which may contribute to cachexia pathophysiology. However, underlying contributors to decreased in vivo cardiac contractility are not well understood. In this study, we sought to distinguish heart-intrinsic changes from systemic factors contributing to cachexia-associated cardiac dysfunction. We hypothesized that isolated heart and cardiac myocyte functional deficits underlie in vivo contractile dysfunction. To test this hypothesis, isolated heart and cardiac myocyte function was measured in the colon-26 adenocarcinoma murine model of cachexia. Ex vivo perfused hearts from cachectic animals exhibited marked contraction and relaxation deficits during basal and pacing conditions. Isolated myocytes displayed significantly decreased peak contraction and relaxation rates, which was accompanied by decreased peak calcium and decay rates. This study uncovers significant organ and cellular-level functional deficits in cachectic hearts outside of the catabolic in vivo environment, which is explained in part by impaired calcium cycling. These data provide insight into physiological mechanisms of cardiomyopathy in cachexia, which is critical for the ultimate development of effective treatments for patients.

Role of plakophilin-2 expression on exercise-related progression of arrhythmogenic right ventricular cardiomyopathy: a translational study.

Exercise increases arrhythmia risk and cardiomyopathy progression in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients, but the mechanisms remain unknown. We investigated transcriptomic changes caused by endurance training in mice deficient in plakophilin-2 (PKP2cKO), a desmosomal protein important for intercalated disc formation, commonly mutated in ARVC and controls. Exercise alone caused transcriptional downregulation of genes coding intercalated disk proteins. The changes converged with those in sedentary and in exercised PKP2cKO mice. PKP2 loss caused cardiac contractile deficit, decreased muscle mass and increased functional/transcriptomic signatures of apoptosis, despite increased fractional shortening and calcium transient amplitude in single myocytes. Exercise accelerated cardiac dysfunction, an effect dampened by pre-training animals prior to PKP2-KO. Consistent with PKP2-dependent muscle mass deficit, cardiac dimensions in human athletes carrying PKP2 mutations were reduced, compared to matched controls. We speculate that exercise challenges a cardiomyocyte "desmosomal reserve" which, if impaired genetically (e.g., PKP2 loss), accelerates progression of cardiomyopathy.