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Pregnant individuals with obesity (body mass index, BMI ≥ 30 kg/m 2 ) are more likely to experience prolonged labor and have double the risk of cesarean compared with individuals with normal weight (BMI < 25 kg/m 2 ). The aim of this study was to evaluate whether obesity in pregnancy is associated with reduced spontaneous and oxytocin-stimulated myometrial contractile activity using ex vivo preparations. We also assessed the relationship between maternal BMI and the expression of oxytocin (OXTR) and prostaglandin (FP) receptors in the myometrial tissue. We enrolled 73 individuals with a singleton gestation undergoing scheduled cesarean delivery at term in a prospective cohort study. This included 49 individuals with a pre-pregnancy BMI ≥ 30 kg/m 2 and 24 with BMI < 25.0 kg/m 2 . After delivery, a small strip of myometrium was excised from the upper edge of the hysterotomy. Baseline spontaneous and oxytocin stimulated myometrial contractile activity was measured using ex vivo preparations. Additionally, expression of oxytocin and prostaglandin receptors from myometrial samples were compared using qRT-PCR and western blot techniques. Spontaneous and oxytocin-stimulated contraction frequency, duration, and force were not significantly different in myometrial samples from the obese and normal-weight individuals. Myometrial OXTR gene and protein expression was also similar in the two groups. While FP gene expression was lower in the myometrial samples from the obese group, protein expression did not differ. These data help to address an important knowledge gap related to the biological mechanisms underlying the association between maternal obesity and dysfunctional labor., (© 2024. The Author(s).)

Background: Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. Signaling pathways that link genetic sequence variants to clinically overt HCM and progression to severe forms of HCM remain unknown., Objectives: The purpose of this study was to identify signaling pathways that are differentially regulated in HCM, using proteomic profiling of human myocardium, confirmed with transcriptomic profiling., Methods: In this multicenter case-control study, myocardial samples were obtained from cases with HCM and control subjects with nonfailing hearts. Proteomic profiling of 7,289 proteins from myocardial samples was performed using the SomaScan assay (SomaLogic). Pathway analysis of differentially expressed proteins was performed, using a false discovery rate <0.05. Pathway analysis of proteins whose concentrations correlated with clinical indicators of severe HCM (eg, reduced left ventricular ejection fraction, atrial fibrillation, and ventricular tachyarrhythmias) was also executed. Confirmatory analysis of differentially expressed genes was performed using myocardial transcriptomic profiling., Results: The study included 99 HCM cases and 15 control subjects. Pathway analysis of differentially expressed proteins revealed dysregulation of the Ras-mitogen-activated protein kinase, ubiquitin-mediated proteolysis, angiogenesis-related (eg, hypoxia-inducible factor-1, vascular endothelial growth factor), and Hippo pathways. Pathways known to be dysregulated in HCM, including metabolic, inflammatory, and extracellular matrix pathways, were also dysregulated. Pathway analysis of proteins associated with clinical indicators of severe HCM and of differentially expressed genes supported these findings., Conclusions: The present study represents the most comprehensive (>7,000 proteins) and largest-scale (n = 99 HCM cases) proteomic profiling of human HCM myocardium to date. Proteomic profiling and confirmatory transcriptomic profiling elucidate dysregulation of both newly recognized (eg, Ras-mitogen-activated protein kinase) and known pathways associated with pathogenesis and progression to severe forms of HCM., Competing Interests: Funding Support and Author Disclosures This work was supported by the National Institutes of Health (R01 HL157216 and R01 HL168382 to Dr Shimada, UL1 TR001873 to Dr Reilly, K24 HL107643 to Dr Reilly, K24 AG036778 to Dr Maurer, R01 HL170132 to Dr Topkara, R01 HL131872 to Dr Ferrari, and T32 HL007854 to Dr Lumish), the American Heart Association (2 National Clinical and Population Research Awards, 1 Career Development Award, and 1 Transformational Project Award to Dr Shimada), Korea Institute of Oriental Medicine (W22005 to Dr Shimada), Feldstein Medical Foundation (to Dr Shimada), Columbia University Irving Medical Center Precision Medicine Pilot Award (to Dr Shimada), and Columbia University Irving Medical Center Marjorie and Lewis Katz Cardiovascular Research Prize (to Dr Shimada). The funding organizations did not have any role in the study design, collection, analysis, or interpretation of data, in writing of the manuscript, or in the decision to submit the paper for publication. The researchers were independent from the funding organizations. Dr Maurer has received consulting income from Akcea, Alnylam, Eidos Therapeutics, Pfizer, Prothena, Novo Nordisk, and Intellia. Dr Shimada has received research funding from Bristol Myers Squibb; and has received consulting income from Bristol Myers Squibb and Moderna Japan. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Heart muscle has the unique property that it can never rest; all cardiomyocytes contract with each heartbeat which requires a complex control mechanism to regulate cardiac output to physiological requirements. Changes in calcium concentration regulate the thin filament activation. A separate but linked mechanism regulates the thick filament activation, which frees sufficient myosin heads to bind the thin filament, thereby producing the required force. Thick filaments contain additional nonmyosin proteins, myosin-binding protein C and titin, the latter being the protein that transmits applied tension to the thick filament. How these three proteins interact to control thick filament activation is poorly understood. Here, we show using 3-D image reconstruction of frozen-hydrated human cardiac muscle myofibrils lacking exogenous drugs that the thick filament is structured to provide three levels of myosin activation corresponding to the three crowns of myosin heads in each 429Å repeat. In one crown, the myosin heads are almost completely activated and disordered. In another crown, many myosin heads are inactive, ordered into a structure called the interacting heads motif. At the third crown, the myosin heads are ordered into the interacting heads motif, but the stability of that motif is affected by myosin-binding protein C. We think that this hierarchy of control explains many of the effects of length-dependent activation as well as stretch activation in cardiac muscle control., Competing Interests: Competing interests statement:J.R.P. provides consulting to Kate Therapeutics, but such work is unrelated to the content of this article.

Heart failure (HF) presents a significant clinical challenge, with current treatments mainly easing symptoms without stopping disease progression. The targeting of calcium (Ca 2+ ) regulation is emerging as a key area for innovative HF treatments that could significantly alter disease outcomes and enhance cardiac function. In this review, we aim to explore the implications of altered Ca 2+ sensitivity, a key determinant of cardiac muscle force, in HF, including its roles during systole and diastole and its association with different HF types-HF with preserved and reduced ejection fraction (HFpEF and HFrEF, respectively). We further highlight the role of the two rate constants k on (Ca 2+ binding to Troponin C) and k off (its dissociation) to fully comprehend how changes in Ca 2+ sensitivity impact heart function. Additionally, we examine how increased Ca 2+ sensitivity, while boosting systolic function, also presents diastolic risks, potentially leading to arrhythmias and sudden cardiac death. This suggests that strategies aimed at moderating myofilament Ca 2+ sensitivity could revolutionize anti-arrhythmic approaches, reshaping the HF treatment landscape. In conclusion, we emphasize the need for precision in therapeutic approaches targeting Ca 2+ sensitivity and call for comprehensive research into the complex interactions between Ca 2+ regulation, myofilament sensitivity, and their clinical manifestations in HF.

Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca 2+ release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female hearts exhibit lower mito-[Ca 2+ ] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca 2+ accumulation, thereby reducing ROS production and stress-induced intracellular Ca 2+ mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca 2+ ] in female rat VCMs challenged with β-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca 2+ uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca 2+ release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca 2+ mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca 2+ ] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca 2+ release. We conclude that sexual dimorphism in mito-Ca 2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females., (© 2023. The Author(s).)

Background: Mitral valve (MV) elongation is a primary hypertrophic cardiomyopathy (HCM) phenotype and contributes to obstruction. The residual MV leaflet that protrudes past the coaptation point is especially susceptible to flow-drag and systolic anterior motion. Histopathological features of MVs in obstructive hypertrophic cardiomyopathy (OHCM), and of residual leaflets specifically, are unknown., Objectives: The purpose of this study was to characterize gross, structural, and cellular histopathologic features of MV residual leaflets in OHCM. On a cellular-level, we assessed for developmental dysregulation of epicardium-derived cell (EPDC) differentiation, adaptive endocardial-to-mesenchymal transition and valvular interstitial cell proliferation, and genetically-driven persistence of cardiomyocytes in the valve., Methods: Structural and immunohistochemical staining were performed on 22 residual leaflets excised as ancillary procedures during myectomy, and compared with 11 control leaflets from deceased patients with normal hearts. Structural components were assessed with hematoxylin and eosin, trichrome, and elastic stains. We stained for EPDCs, EPDC paracrine signaling, valvular interstitial cells, endocardial-to-mesenchymal transition, and cardiomyocytes., Results: The residual leaflet was always at A2 segment and attached by slack, elongated and curlicued, myxoid chords. MV residual leaflets in OHCM were structurally disorganized, with expanded spongiosa and increased, fragmented elastic fibers compared with control leading edges. The internal collagenous fibrosa was attenuated and there was collagenous tissue overlying valve surfaces in HCM, with an overall trend toward decreased leaflet thickness (1.09 vs 1.47 mm, P = 0.08). No markers of primary cellular processes were identified., Conclusions: MV residual leaflets in HCM were characterized by histologic findings that were likely secondary to chronic hemodynamic stress and may further increase susceptibility to systolic anterior motion.

Background: The left and right ventricles of the human heart differ in embryology, shape, thickness, and function. Ventricular dyssynchrony often occurs in cases of heart failure. Our objectives were to assess whether differences in contractile properties exist between the left and right ventricles and to evaluate signs of left/right ventricular mechanical synchrony in isolated healthy and diseased human myocardium., Methods: Myocardial left and right ventricular trabeculae were dissected from nonfailing and end-stage failing human hearts. Baseline contractile force and contraction/relaxation kinetics of the left ventricle were compared to those of the right ventricle in the nonfailing group (n=41) and in the failing group (n=29). Correlation analysis was performed to assess the mechanical synchrony between left and right ventricular myocardium isolated from the same heart, in nonfailing (n=41) and failing hearts (n=29)., Results: The failing right ventricular myocardium showed significantly higher developed force (Fdev; P =0.001; d=0.98), prolonged time to peak ( P <0.001; d=1.14), and higher rate of force development ( P =0.002; d=0.89) and force decline ( P =0.003; d=0.82) compared to corresponding left ventricular myocardium. In healthy myocardium, a strong positive relationship was present between the left and right ventricles in time to peak (r=0.58, P <0.001) and maximal kinetic rate of contraction (r=0.63, P <0.001). These coefficients were much weaker, often nearly absent, in failing myocardium., Conclusions: At the level of isolated cardiac trabeculae, contractile performance, specifically of contractile kinetics, is correlated in the nonfailing myocardium between the left and right ventricles' but this correlation is significantly weaker, or even absent, in end-stage heart failure, suggesting an interventricular mechanical dyssynchrony.

Troponin I (TnI) is a key regulator of cardiac contraction and relaxation with TnI Ser-23/24 phosphorylation serving as a myofilament mechanism to modulate cardiac function. Basal cardiac TnI Ser-23/24 phosphorylation is high such that both increased and decreased TnI phosphorylation may modulate cardiac function. While the effects of increasing TnI Ser-23/24 phosphorylation on heart function are well established, the effects of decreasing TnI Ser-23/24 phosphorylation are not clear. To understand the in vivo role of decreased TnI Ser-23/24 phosphorylation, mice expressing TnI with Ser-23/24 mutated to alanine (TnI S23/24A) that lack the ability to be phosphorylated at these residues were subjected to echocardiography and pressure-volume hemodynamic measurements in the absence or presence of physiological (pacing increasing heart rate or adrenergic stimulation) or pathological (transverse aortic constriction (TAC)) stress. In the absence of pathological stress, the lack of TnI Ser-23/24 phosphorylation impaired systolic and diastolic function. TnI S23/24A mice also had an impaired systolic and diastolic response upon stimulation increased heart rate and an impaired adrenergic response upon dobutamine infusion. Following pathological cardiac stress induced by TAC, TnI S23/24A mice had a greater increase in ventricular mass, worse diastolic function, and impaired systolic and diastolic function upon increasing heart rate. These findings demonstrate that mice lacking the ability to phosphorylate TnI at Ser-23/24 have impaired in vivo systolic and diastolic cardiac function, a blunted cardiac reserve and a worse response to pathological stress supporting decreased TnI Ser23/24 phosphorylation is a modulator of these processes in vivo., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Mitochondrial-associated membranes (MAMs) are known to modulate organellar and cellular functions and can subsequently affect pathophysiology including myocardial ischemia-reperfusion (IR) injury. Thus, identifying molecular targets in MAMs that regulate the outcome of IR injury will hold a key to efficient therapeutics. Here, we found chloride intracellular channel protein (CLIC4) presence in MAMs of cardiomyocytes and demonstrate its role in modulating ER and mitochondrial calcium homeostasis under physiological and pathological conditions. In a murine model, loss of CLIC4 increased myocardial infarction and substantially reduced cardiac function after IR injury. CLIC4 null cardiomyocytes showed increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation injury in comparison to wild-type cardiomyocytes. Overall, our results indicate that MAM-CLIC4 is a key mediator of cellular response to IR injury and therefore may have a potential implication on other pathophysiological processes.

For patients with heart failure, myocardial ATP level can be reduced to one-half of that observed in healthy controls. This marked reduction (from ≈8 mM in healthy controls to as low as 3-4 mM in heart failure) has been suggested to contribute to impaired myocardial contraction and to the decreased pump function characteristic of heart failure. However, in vitro measures of maximum myofilament force generation, maximum shortening velocity, and the actomyosin ATPase activity show effective K M values for MgATP ranging from ≈10 μM to 150 μM, well below the intracellular ATP level in heart failure. Thus, it is not clear that the fall of myocardial ATP observed in heart failure is sufficient to impair the function of the contractile proteins. Therefore, we tested the effect of low MgATP levels on myocardial contraction using demembranated cardiac muscle preparations that were exposed to MgATP levels typical of the range found in non-failing and failing hearts. Consistent with previous studies, we found that a 50% reduction in MgATP level (from 8 mM to 4 mM) did not reduce maximum force generation or maximum velocity of shortening. However, we found that a 50% reduction in MgATP level caused a 20%-25% reduction in maximal power generation (measured during muscle shortening against a load) and a 20% slowing of cross-bridge cycling kinetics. These results suggest that the decreased cellular ATP level occurring in heart failure contributes to the impaired pump function of the failing heart. Since the ATP-myosin ATPase dissociation constant is estimated to be submillimolar, these findings also suggest that MgATP concentration affects cross-bridge dynamics through a mechanism that is more complex than through the direct dependence of MgATP concentration on myosin ATPase activity. Finally, these studies suggest that therapies targeted to increase adenine nucleotide pool levels in cardiomyocytes might be beneficial for treating heart failure., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Background Because body mass index (BMI) is generally used clinically to define obesity and to estimate body adiposity, BMI likely is positively correlated with epicardial adipose tissue (EAT) level. Based on echocardiography, previous outcomes on this matter have varied from almost absent to rather strong correlations between BMI and EAT. The purpose of our study was to unambiguously examine EAT content and determine if correlations exist between EAT content and BMI, cause of heart failure, or contractile force. Methods and Results We qualitatively scored 150 human hearts ex vivo on EAT distribution. From each heart, multiple photographs of the heart were taken, and both atrial and ventricular adipose tissue levels were semiquantitatively scored. Main findings include a generally higher EAT content on nonfailing hearts compared with end-stage failing hearts (atrial adipose tissue level 5.70±0.13 vs. 5.00±0.12, P <0.001; ventricular adipose tissue level 5.14±0.16 vs. 4.57±0.12, P =0.0048). The results also suggest that EAT quantity is not strongly correlated with BMI in nonfailing (atrial adipose tissue level r =0.069, ventricular adipose tissue level r =0.14) or failing (atrial adipose tissue level r =-0.022, ventricular adipose tissue level r =0.051) hearts. Atrial EAT is closely correlated with ventricular EAT in both nonfailing ( r =0.92, P <0.001) and failing ( r =0.87, P <0.001) hearts. Conclusions EAT volume appears to be inversely proportional to severity of or length of time with heart failure based on our findings. Based on a lack of correlation with BMI, it is incorrect to assume high EAT volume given high body fat percentage.

Ubiquitin carboxyl-terminal esterase L1 (UCHL1) has been thought to be a neuron specific protein and shown to play critical roles in Parkinson's Disease and stroke via de-ubiquiting and stabilizing key pathological proteins, such as α-synuclein. In the present study, we found that UCHL1 was significantly increased in both mouse and human cardiomyocytes following myocardial infarction (MI). When LDN-57444, a pharmacological inhibitor of UCHL1, was used to treat mice subjected to MI surgery, we found that administration of LDN-57444 compromised cardiac function when compared with vehicle treated hearts, suggesting a potential protective role of UCHL1 in response to MI. When UCHL1 was knockout by CRISPR/Cas 9 gene editing technique in human induced pluripotent stem cells (hiPSCs), we found that cardiomyocytes derived from UCHL1-/- hiPSCs were more susceptible to hypoxia/re-oxygenation induced injury as compared to wild type cardiomyocytes. To study the potential targets of UCHL1, a BioID based proximity labeling approach followed by mass spectrum analysis was performed. The result suggested that UCHL1 could bind to and stabilize HIF-1α following MI. Indeed, expression of HIF-1α was lower in UCHL1-/- cells as determined by Western blotting and HIF-1α target genes were also suppressed in UCHL1-/- cells as quantified by real time RT-PCR. Recombinant UCHL1 (rUCHL1) protein was purified by E. Coli fermentation and intraperitoneally (I.P.) delivered to mice. We found that administration of rUCHL1 could significantly preserve cardiac function following MI as compared to control group. Finally, adeno associated virus mediated cardiac specific UCHL1 delivery (AAV9-cTNT-m-UCHL1) was performed in neonatal mice. UCHL1 overexpressing hearts were more resistant to MI injury as compare to the hearts infected with control virus. In summary, our data revealed a novel protective role of UCHL1 on MI via stabilizing HIF-1α and promoting HIF-1α signaling., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Phosphorylation and acetylation of sarcomeric proteins are important for fine-tuning myocardial contractility. Here, we used bottom-up proteomics and label-free quantification to identify novel post-translational modifications (PTMs) on β-myosin heavy chain (β-MHC) in normal and failing human heart tissues. We report six acetylated lysines and two phosphorylated residues: K34-Ac, K58-Ac, S210-P, K213-Ac, T215-P, K429-Ac, K951-Ac, and K1195-Ac. K951-Ac was significantly reduced in both ischemic and nonischemic failing hearts compared to nondiseased hearts. Molecular dynamics (MD) simulations show that K951-Ac may impact stability of thick filament tail interactions and ultimately myosin head positioning. K58-Ac altered the solvent-exposed SH3 domain surface - known for protein-protein interactions - but did not appreciably change motor domain conformation or dynamics under conditions studied. Together, K213-Ac/T215-P altered loop 1's structure and dynamics - known to regulate ADP-release, ATPase activity, and sliding velocity. Our study suggests that β-MHC acetylation levels may be influenced more by the PTM location than the type of heart disease since less protected acetylation sites are reduced in both heart failure groups. Additionally, these PTMs have potential to modulate interactions between β-MHC and other regulatory sarcomeric proteins, ADP-release rate of myosin, flexibility of the S2 region, and cardiac myofilament contractility in normal and failing hearts., Competing Interests: ML, MC, AW, MG, HH, RS, EB, JJ, BW, PC, PJ, MR, BB, JP, MP No competing interests declared, (© 2022, Landim-Vieira et al.)

The relationship between hypothyroidism and the occurrence and progression of heart failure (HF) has had increased interest over the past years. The low T3 syndrome, a reduced T3 in the presence of normal thyroid stimulating hormone (TSH), and free T4 concentration, is a strong predictor of all-cause mortality in HF patients. Still, the impact of hypothyroidism on the contractile properties of failing human myocardium is unknown. Our study aimed to investigate that impact using ex-vivo assessment of force and kinetics of contraction/relaxation in left ventricular intact human myocardial muscle preparations. Trabeculae were dissected from non-failing (NF; n = 9), failing with no hypothyroidism (FNH; n = 9), and failing with hypothyroidism (FH; n = 9) hearts. Isolated muscle preparations were transferred into a custom-made setup where baseline conditions as well as the three main physiological modulators that regulate the contractile strength, length-dependent and frequency-dependent activation, as well as β-adrenergic stimulation, were assessed under near-physiological conditions. Hypothyroidism did not show any additional significant impact on the contractile properties different from the recognized alterations usually detected in such parameters in any end-stage failing heart without thyroid dysfunction. Clinical information for FH patients in our study revealed they were all receiving levothyroxine. Absence of any difference between failing hearts with or without hypothyroidism, may possibly be due to the profound effects of the advanced stage of heart failure that concealed any changes between the groups. Still, we cannot exclude the possibility of differences that may have been present at earlier stages. The effects of THs supplementation such as levothyroxine on contractile force and kinetic parameters of failing human myocardium require further investigation to explore its full potential in improving cardiovascular performance and cardiovascular outcomes of HF associated with hypothyroidism., Competing Interests: The authors have declared that no competing interests exist.

Cardiovascular disease (CVD) and stroke affect over 92 million Americans and account for nearly 1 out of 3 deaths in the US. The use of animal models in cardiovascular research has led to considerable advances in treatment and in our understanding of the pathophysiology of many CVDs. Still, animals may not fully recapitulate human disease states; species differences have long been postulated to be one of the main reasons for a failure of translation between animals and humans in drug discovery and development. Indeed, it has become increasingly clear over the past few decades that to answer certain biomedical questions, like the physiological mechanisms that go awry in many human CVDs, animal tissues may not always be the best option to use. While human cardiac tissue has long been used for laboratory research, published findings often contradict each other, leading to difficulties in interpretation. Current difficulties in utilizing human cardiac tissue include differences in acquisition time, varying tissue procurement protocols, and the struggle to define a human "control" sample. With the tremendous emphasis on translational research that continues to grow, research studies using human tissues are becoming more common. This mini review will discuss advantages, disadvantages, and considerations of using human cardiac tissue in the study of CVDs, paying specific attention to the study of phosphoproteins., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Huang and Janssen.)

Zonula occludens-1 (ZO-1) is an intracellular scaffolding protein that orchestrates the anchoring of membrane proteins to the cytoskeleton in epithelial and specialized tissue including the heart. There is clear evidence to support the central role of intracellular auxiliary proteins in arrhythmogenesis and previous studies have found altered ZO-1 expression associated with atrioventricular conduction abnormalities. Here, using human cardiac tissues, we identified all three isoforms of ZO-1, canonical (Transcript Variant 1, TV1 ), CRA&#95;e (Transcript Variant 4, TV4 ), and an additionally expressed (Transcript Variant 3, TV3 ) in non-failing myocardium. To investigate the role of ZO-1 on ventricular arrhythmogenesis, we generated a haploinsufficient ZO-1 mouse model (ZO-1 +/- ). ZO-1 +/- mice exhibited dysregulated connexin-43 protein expression and localization at the intercalated disc. While ZO-1 +/- mice did not display abnormal cardiac function at baseline, adrenergic challenge resulted in rhythm abnormalities, including premature ventricular contractions and bigeminy. At baseline, ventricular myocytes from the ZO-1 +/- mice displayed prolonged action potential duration and spontaneous depolarizations, with ZO-1 +/- cells displaying frequent unsolicited (non-paced) diastolic depolarizations leading to spontaneous activity with multiple early afterdepolarizations (EADs). Mechanistically, ZO-1 deficient myocytes displayed a reduction in sodium current density ( I Na ) and an increased sensitivity to isoproterenol stimulation. Further, ZO-1 deficient myocytes displayed remodeling in I Ca current, likely a compensatory change. Taken together, our data suggest that ZO-1 deficiency results in myocardial substrate susceptible to triggered arrhythmias.

Memantine is a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist that was initially indicated for the treatment of moderate to severe Alzheimer's disease. It is now also considered for a variety of other pathologies in which activation of NMDA receptors apparently contributes to the pathogenesis and progression of disease. In addition to the central nervous system (CNS), NMDA receptors can be found in non-neuronal cells and tissues that recently have become an interesting research focus. Some studies have shown that glutamate signaling plays a role in cell transformation and cancer progression. In addition, these receptors may play a role in cardiovascular disorders. In this review, we focus on the most recent findings for memantine with respect to its pharmacological effects in a range of diseases, including inflammatory disorders, cardiovascular diseases, cancer, neuropathy, as well as retinopathy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

It is widely assumed that synthesis of membrane proteins, particularly in the heart, follows the classical secretory pathway with mRNA translation occurring in perinuclear regions followed by protein trafficking to sites of deployment. However, this view is based on studies conducted in less-specialized cells, and has not been experimentally addressed in cardiac myocytes. Therefore, we undertook direct experimental investigation of protein synthesis in cardiac tissue and isolated myocytes using single-molecule visualization techniques and a novel proximity-ligated in situ hybridization approach for visualizing ribosome-associated mRNA molecules for a specific protein species, indicative of translation sites. We identify here, for the first time, that the molecular machinery for membrane protein synthesis occurs throughout the cardiac myocyte, and enables distributed synthesis of membrane proteins within sub-cellular niches where the synthesized protein functions using local mRNA pools trafficked, in part, by microtubules. We also observed cell-wide distribution of membrane protein mRNA in myocardial tissue from both non-failing and hypertrophied (failing) human hearts, demonstrating an evolutionarily conserved distributed mechanism from mouse to human. Our results identify previously unanticipated aspects of local control of cardiac myocyte biology and highlight local protein synthesis in cardiac myocytes as an important potential determinant of the heart's biology in health and disease., (© 2021. The Author(s).)

Heart failure (HF) is frequently accompanied with the sinoatrial node (SAN) dysfunction, which causes tachy-brady arrhythmias and increased mortality. MicroRNA (miR) alterations are associated with HF progression. However, the transcriptome of HF human SAN, and its role in HF-associated remodeling of ion channels, transporters, and receptors responsible for SAN automaticity and conduction impairments is unknown. We conducted comprehensive high-throughput transcriptomic analysis of pure human SAN primary pacemaker tissue and neighboring right atrial tissue from human transplanted HF hearts (n = 10) and non-failing (nHF) donor hearts (n = 9), using next-generation sequencing. Overall, 47 miRs and 832 mRNAs related to multiple signaling pathways, including cardiac diseases, tachy-brady arrhythmias and fibrosis, were significantly altered in HF SAN. Of the altered miRs, 27 are predicted to regulate mRNAs of major ion channels and neurotransmitter receptors which are involved in SAN automaticity (e.g. HCN1, HCN4, SLC8A1) and intranodal conduction (e.g. SCN5A, SCN8A) or both (e.g. KCNJ3, KCNJ5). Luciferase reporter assays were used to validate interactions of miRs with predicted mRNA targets. In conclusion, our study provides a profile of altered miRs in HF human SAN, and a novel transcriptome blueprint to identify molecular targets for SAN dysfunction and arrhythmia treatments in HF., (© 2021. The Author(s).)

Aging is associated with chronic oxidative stress and inflammation that affect tissue repair and regeneration capacity. MG53 is a TRIM family protein that facilitates repair of cell membrane injury in a redox-dependent manner. Here, we demonstrate that the expression of MG53 was reduced in failing human hearts and aged mouse hearts, concomitant with elevated NF-κB activation. We evaluated the safety and efficacy of longitudinal, systemic administration of recombinant human MG53 (rhMG53) protein in aged mice. Echocardiography and pressure-volume loop measurements revealed beneficial effects of rhMG53 treatment in improving heart function of aged mice. Biochemical and histological studies demonstrated that the cardioprotective effects of rhMG53 are linked to suppression of NF-κB-mediated inflammation, reducing apoptotic cell death and oxidative stress in the aged heart. Repetitive administration of rhMG53 in aged mice did not have adverse effects on major vital organ functions. These findings support the therapeutic value of rhMG53 in treating age-related decline in cardiac function.

Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5'-monophospho-(CMP)-N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome-linked muscular dystrophy (mdx) model for DMD. Cmah -/- mdx mice can be induced to develop anti-Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah -/- mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Up to 50% of the adult human sinoatrial node (SAN) is composed of dense connective tissue. Cardiac diseases including heart failure (HF) may increase fibrosis within the SAN pacemaker complex, leading to impaired automaticity and conduction of electric activity to the atria. Unlike the role of cardiac fibroblasts in pathologic fibrotic remodeling and tissue repair, nothing is known about fibroblasts that maintain the inherently fibrotic SAN environment., Methods: Intact SAN pacemaker complex was dissected from cardioplegically arrested explanted nonfailing hearts (non-HF; n=22; 48.7±3.1 years of age) and human failing hearts (n=16; 54.9±2.6 years of age). Connective tissue content was quantified from Masson trichrome-stained head-center and center-tail SAN sections. Expression of extracellular matrix proteins, including collagens 1 and 3A1, CILP1 (cartilage intermediate layer protein 1), and POSTN (periostin), and fibroblast and myofibroblast numbers were quantified by in situ and in vitro immunolabeling. Fibroblasts from the central intramural SAN pacemaker compartment (≈10×5×2 mm 3 ) and right atria were isolated, cultured, passaged once, and treated ± transforming growth factor β1 and subjected to comprehensive high-throughput next-generation sequencing of whole transcriptome, microRNA, and proteomic analyses., Results: Intranodal fibrotic content was significantly higher in SAN pacemaker complex from HF versus non-HF hearts (57.7±2.6% versus 44.0±1.2%; P <0.0001). Proliferating phosphorylated histone 3 + /vimentin + /CD31 - (cluster of differentiation 31) fibroblasts were higher in HF SAN. Vimentin + /α-smooth muscle actin + /CD31 - myofibroblasts along with increased interstitial POSTN expression were found only in HF SAN. RNA sequencing and proteomic analyses identified unique differences in mRNA, long noncoding RNA, microRNA, and proteomic profiles between non-HF and HF SAN and right atria fibroblasts and transforming growth factor β1-induced myofibroblasts. Specifically, proteins and signaling pathways associated with extracellular matrix flexibility, stiffness, focal adhesion, and metabolism were altered in HF SAN fibroblasts compared with non-HF SAN., Conclusions: This study revealed increased SAN-specific fibrosis with presence of myofibroblasts, CILP1, and POSTN-positive interstitial fibrosis only in HF versus non-HF human hearts. Comprehensive proteotranscriptomic profiles of SAN fibroblasts identified upregulation of genes and proteins promoting stiffer SAN extracellular matrix in HF hearts. Fibroblast-specific profiles generated by our proteotranscriptomic analyses of the human SAN provide a comprehensive framework for future studies to investigate the role of SAN-specific fibrosis in cardiac rhythm regulation and arrhythmias.

The two main phases of the mammalian cardiac cycle are contraction and relaxation; however, whether there is a connection between them in humans is not well understood. Routine exercise has been shown to improve cardiac function, morphology, and molecular signatures. Likewise, the acute and chronic changes that occur in the heart in response to injury, disease, and stress are well characterized, albeit not fully understood. In this study, we investigated how exercise and myocardial injury affect contraction-relaxation coupling. We retrospectively analyzed the correlation between the maximal speed of contraction and the maximal speed of relaxation of canine myocardium after receiving surgically induced myocardial infarction, followed by either sedentary recovery or exercise training for 10-12 wk. We used isolated right ventricular trabeculae, which were electrically paced at different lengths, frequencies, and with increasing β-adrenoceptor stimulation. In all conditions, contraction and relaxation were linearly correlated, irrespective of injury or training history. Based on these results and the available literature, we posit that contraction-relaxation coupling is a fundamental myocardial property that resides in the structural arrangement of proteins at the level of the sarcomere and that this may be regulated by the actions of cardiac myosin binding protein C (cMyBP-C) on actin and myosin., (© 2021 Fazlollahi et al.)

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C ∆C0-C1f ), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Heart failure (HF) is associated with highly significant morbidity, mortality, and health care costs. Despite the significant advances in therapies and prevention, HF remains associated with poor clinical outcomes. Understanding the contractile force and kinetic changes at the level of cardiac muscle during end-stage HF in consideration of underlying etiology would be beneficial in developing targeted therapies that can help improve cardiac performance., Objective: Investigate the impact of the primary etiology of HF (ischemic or non-ischemic) on left ventricular (LV) human myocardium force and kinetics of contraction and relaxation under near-physiological conditions., Methods and Results: Contractile and kinetic parameters were assessed in LV intact trabeculae isolated from control non-failing (NF; n = 58) and end-stage failing ischemic (FI; n = 16) and non-ischemic (FNI; n = 38) human myocardium under baseline conditions, length-dependent activation, frequency-dependent activation, and response to the β-adrenergic stimulation. At baseline, there were no significant differences in contractile force between the three groups; however, kinetics were impaired in failing myocardium with significant slowing down of relaxation kinetics in FNI compared to NF myocardium. Length-dependent activation was preserved and virtually identical in all groups. Frequency-dependent activation was clearly seen in NF myocardium (positive force frequency relationship [FFR]), while significantly impaired in both FI and FNI myocardium (negative FFR). Likewise, β-adrenergic regulation of contraction was significantly impaired in both HF groups., Conclusions: End-stage failing myocardium exhibited impaired kinetics under baseline conditions as well as with the three contractile regulatory mechanisms. The pattern of these kinetic impairments in relation to NF myocardium was mainly impacted by etiology with a marked slowing down of kinetics in FNI myocardium. These findings suggest that not only force development, but also kinetics should be considered as a therapeutic target for improving cardiac performance and thus treatment of HF., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

[Figure: see text].

Regulation of gene expression plays a fundamental role in cardiac stress-responses. Modification of coding transcripts by adenosine methylation (m 6 A) has recently emerged as a critical post-transcriptional mechanism underlying heart disease. Thousands of mammalian mRNAs are known to be m 6 A-modified, suggesting that remodeling of the m 6 A landscape may play an important role in cardiac pathophysiology. Here we found an increase in m 6 A content in human heart failure samples. We then adopted genome-wide analysis to define all m 6 A-regulated sites in human failing compared to non-failing hearts and identified targeted transcripts involved in histone modification as enriched in heart failure. Further, we compared all m 6 A sites regulated in human hearts with the ones occurring in isolated rat hypertrophic cardiomyocytes to define cardiomyocyte-specific m 6 A events conserved across species. Our results identified 38 shared transcripts targeted by m 6 A during stress conditions, and 11 events that are unique to unstressed cardiomyocytes. Of these, further evaluation of select mRNA and protein abundances demonstrates the potential impact of m 6 A on post-transcriptional regulation of gene expression in the heart., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aims: Duchenne muscular dystrophy (DMD) is an X-linked inherited disease due to dystrophin deficiency causing skeletal and cardiac muscle dysfunction. Affected patients lose ambulation by age 12 and usually die in the second to third decades of life from cardiac and respiratory failure. Symptomatic treatment includes the use of anti-inflammatory corticosteroids, which are associated with side effects including weight gain, osteoporosis, and increased risk of cardiovascular disease. Novel treatment options include blockade of the renin-angiotensin-aldosterone system, because angiotensin as well as aldosterone contribute to persistent inflammation and fibrosis, and aldosterone blockade represents an efficacious anti-fibrotic approach in cardiac failure. Recent preclinical findings enabled successful clinical testing of a combination of steroidal mineralocorticoid receptor antagonists (MRAs) and angiotensin converting enzyme inhibitors in DMD boys. The efficacy of MRAs alone on dystrophic skeletal muscle and heart has not been investigated. Here, we tested efficacy of the novel non-steroidal MRA finerenone as a monotherapy in a preclinical DMD model., Methods and Results: The dystrophin-deficient, utrophin haploinsufficient mouse model of DMD was treated with finerenone and compared with untreated dystrophic and wild-type controls. Grip strength, electrocardiography, cardiac magnetic resonance imaging, muscle force measurements, histological quantification, and gene expression studies were performed. Finerenone treatment alone resulted in significant improvements in clinically relevant functional parameters in both skeletal muscle and heart. Normalized grip strength in rested dystrophic mice treated with finerenone (40.3 ± 1.0 mN/g) was significantly higher (P = 0.0182) compared with untreated dystrophic mice (35.2 ± 1.5 mN/g). Fatigued finerenone-treated dystrophic mice showed an even greater relative improvement (P = 0.0003) in normalized grip strength (37.5 ± 1.1 mN/g) compared with untreated mice (29.7 ± 1.1 mN/g). Finerenone treatment also led to significantly lower (P = 0.0075) susceptibility to limb muscle damage characteristic of DMD measured during a contraction-induced injury protocol. Normalized limb muscle force after five lengthening contractions resulted in retention of 71 ± 7% of baseline force in finerenone-treated compared with only 51 ± 4% in untreated dystrophic mice. Finerenone treatment also prevented significant reductions in myocardial strain rate (P = 0.0409), the earliest sign of DMD cardiomyopathy. Moreover, treatment with finerenone led to very specific cardiac gene expression changes in clock genes that might modify cardiac pathophysiology in this DMD model., Conclusions: Finerenone administered as a monotherapy is disease modifying for both skeletal muscle and heart in a preclinical DMD model. These findings support further evaluation of finerenone in DMD clinical trials., (© 2020 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

Background Atrial fibrillation (AF) driver mechanisms are obscured to clinical multielectrode mapping approaches that provide partial, surface-only visualization of unstable 3-dimensional atrial conduction. We hypothesized that transient modulation of refractoriness by pharmacologic challenge during multielectrode mapping improves visualization of hidden paths of reentrant AF drivers for targeted ablation. Methods and Results Pharmacologic challenge with adenosine was tested in ex vivo human hearts with a history of AF and cardiac diseases by multielectrode and high-resolution subsurface near-infrared optical mapping, integrated with 3-dimensional structural imaging and heart-specific computational simulations. Adenosine challenge was also studied on acutely terminated AF drivers in 10 patients with persistent AF. Ex vivo, adenosine stabilized reentrant driver paths within arrhythmogenic fibrotic hubs and improved visualization of reentrant paths, previously seen as focal or unstable breakthrough activation pattern, for targeted AF ablation. Computational simulations suggested that shortening of atrial refractoriness by adenosine may (1) improve driver stability by annihilating spatially unstable functional blocks and tightening reentrant circuits around fibrotic substrates, thus unmasking the common reentrant path; and (2) destabilize already stable reentrant drivers along fibrotic substrates by accelerating competing fibrillatory wavelets or secondary drivers. In patients with persistent AF, adenosine challenge unmasked hidden common reentry paths (9/15 AF drivers, 41±26% to 68±25% visualization), but worsened visualization of previously visible reentry paths (6/15, 74±14% to 34±12%). AF driver ablation led to acute termination of AF. Conclusions Our ex vivo to in vivo human translational study suggests that transiently altering atrial refractoriness can stabilize reentrant paths and unmask arrhythmogenic hubs to guide targeted AF driver ablation treatment.

Background: Atrial fibrillation (AF) can be maintained by localized intramural reentrant drivers. However, AF driver detection by clinical surface-only multielectrode mapping (MEM) has relied on subjective interpretation of activation maps. We hypothesized that application of machine learning to electrogram frequency spectra may accurately automate driver detection by MEM and add some objectivity to the interpretation of MEM findings., Methods: Temporally and spatially stable single AF drivers were mapped simultaneously in explanted human atria (n=11) by subsurface near-infrared optical mapping (NIOM; 0.3 mm 2 resolution) and 64-electrode MEM (higher density or lower density with 3 and 9 mm 2 resolution, respectively). Unipolar MEM and NIOM recordings were processed by Fourier transform analysis into 28 407 total Fourier spectra. Thirty-five features for machine learning were extracted from each Fourier spectrum., Results: Targeted driver ablation and NIOM activation maps efficiently defined the center and periphery of AF driver preferential tracks and provided validated annotations for driver versus nondriver electrodes in MEM arrays. Compared with analysis of single electrogram frequency features, averaging the features from each of the 8 neighboring electrodes, significantly improved classification of AF driver electrograms. The classification metrics increased when less strict annotation, including driver periphery electrodes, were added to driver center annotation. Notably, f1-score for the binary classification of higher-density catheter data set was significantly higher than that of lower-density catheter (0.81±0.02 versus 0.66±0.04, P <0.05). The trained algorithm correctly highlighted 86% of driver regions with higher density but only 80% with lower-density MEM arrays (81% for lower-density+higher-density arrays together)., Conclusions: The machine learning model pretrained on Fourier spectrum features allows efficient classification of electrograms recordings as AF driver or nondriver compared with the NIOM gold-standard. Future application of NIOM-validated machine learning approach may improve the accuracy of AF driver detection for targeted ablation treatment in patients.

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by the lack of functional dystrophin protein. In muscular dystrophy preclinical research, it is pertinent to analyze the force of the muscles affected by the disease to assess pathology and potential effectiveness of therapeutic interventions. Although muscles function at sub-maximal levels in vivo , maximal tetanic contractions are most commonly used to assess and report muscle function in muscular dystrophy studies. At submaximal activation, the kinetics of contraction and relaxation are heavily impacted by the kinetics of the single twitch. However, maximal tetanic force is often the main, if not sole, outcome measured in most studies, while contractile kinetics are rarely reported. To investigate the effect of muscle disease on twitch contraction kinetics, isolated diaphragm and extensor digitorum longus (EDL) muscles of 10-, 20-week, "het" (dystrophin deficient and utrophin haplo-insufficient), and 52-week mdx (dystrophin deficient) mice were analyzed and compared to wild-type controls. We observed that twitch contractile kinetics are dependent on muscle type, age, and disease state. Specific findings include that diaphragm from wildtype mice has a greater time to 50% relaxation (RT50) than time to peak tension (TTP) compared to the het and mdx dystrophic models, where there is a similar TTP compared to RT50. Diaphragm twitch kinetics remain virtually unchanged with age, while the EDL from het and mdx mice initially has a greater RT50 than TTP, but the TTP increases with age. The difference between EDL contractile kinetics of dystrophic and wildtype mice is more prominent at young age. Differences in kinetics yielded greater statistical significance compared to previously published force measurements, thus, using kinetics as an outcome parameter could potentially allow for use of smaller experimental groups in future study designs. Although this study focused on DMD models, our findings may be applicable to other skeletal muscle conditions and diseases., (Copyright © 2020 Peczkowski, Rastogi, Lowe, Floyd, Schultz, Karaze, Davis, Rafael-Fortney and Janssen.)

We identified ten persons in six consanguineous families with distal arthrogryposis (DA) who had congenital contractures, scoliosis, and short stature. Exome sequencing revealed that each affected person was homozygous for one of two different rare variants (c.470G>T [p.Cys157Phe] or c.469T>C [p.Cys157Arg]) affecting the same residue of myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF). In a seventh family, a c.487G>A (p.Gly163Ser) variant in MYLPF arose de novo in a father, who transmitted it to his son. In an eighth family comprised of seven individuals with dominantly inherited DA, a c.98C>T (p.Ala33Val) variant segregated in all four persons tested. Variants in MYLPF underlie both dominant and recessively inherited DA. Mylpf protein models suggest that the residues associated with dominant DA interact with myosin whereas the residues altered in families with recessive DA only indirectly impair this interaction. Pathological and histological exam of a foot amputated from an affected child revealed complete absence of skeletal muscle (i.e., segmental amyoplasia). To investigate the mechanism for this finding, we generated an animal model for partial MYLPF impairment by knocking out zebrafish mylpfa. The mylpfa mutant had reduced trunk contractile force and complete pectoral fin paralysis, demonstrating that mylpf impairment most severely affects limb movement. mylpfa mutant muscle weakness was most pronounced in an appendicular muscle and was explained by reduced myosin activity and fiber degeneration. Collectively, our findings demonstrate that partial loss of MYLPF function can lead to congenital contractures, likely as a result of degeneration of skeletal muscle in the distal limb., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Bradyarrhythmias are an important cause of mortality in heart failure and previous studies indicate a mechanistic role for electrical remodelling of the key pacemaking ion channel HCN4 in this process. Here we show that, in a mouse model of heart failure in which there is sinus bradycardia, there is upregulation of a microRNA (miR-370-3p), downregulation of the pacemaker ion channel, HCN4, and downregulation of the corresponding ionic current, I f , in the sinus node. In vitro, exogenous miR-370-3p inhibits HCN4 mRNA and causes downregulation of HCN4 protein, downregulation of I f , and bradycardia in the isolated sinus node. In vivo, intraperitoneal injection of an antimiR to miR-370-3p into heart failure mice silences miR-370-3p and restores HCN4 mRNA and protein and I f in the sinus node and blunts the sinus bradycardia. In addition, it partially restores ventricular function and reduces mortality. This represents a novel approach to heart failure treatment.

Skeletal muscle fusion occurs during development, growth, and regeneration. To investigate how muscle fusion compares among different muscle cell types and developmental stages, we studied muscle cell fusion over time in wild-type, myomaker (mymk), and jam2a mutant zebrafish. Using live imaging, we show that embryonic myoblast elongation and fusion correlate tightly with slow muscle cell migration. In wild-type embryos, only fast muscle fibers are multinucleate, consistent with previous work showing that the cell fusion regulator gene mymk is specifically expressed throughout the embryonic fast muscle domain. However, by 3 weeks post-fertilization, slow muscle fibers also become multinucleate. At this late-larval stage, mymk is not expressed in muscle fibers, but is expressed in small cells near muscle fibers. Although previous work showed that both mymk and jam2a are required for embryonic fast muscle cell fusion, we observe that muscle force and function is almost normal in mymk and jam2a mutant embryos, despite the lack of fast muscle multinucleation. We show that genetic requirements change post-embryonically, with jam2a becoming much less important by late-larval stages and mymk now required for muscle fusion and growth in both fast and slow muscle cell types. Correspondingly, adult mymk mutants perform poorly in sprint and endurance tests compared to wild-type and jam2a mutants. We show that adult mymk mutant muscle contains small mononucleate myofibers with average myonuclear domain size equivalent to that in wild type adults. The mymk mutant fibers have decreased Laminin expression and increased numbers of Pax7-positive cells, suggesting that impaired fiber growth and active regeneration contribute to the muscle phenotype. Our findings identify several aspects of muscle fusion that change with time in slow and fast fibers as zebrafish develop beyond embryonic stages., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aims: Heart failure (HF) patients commonly experience symptoms primarily during elevated heart rates, as a result of physical activities or stress. A main determinant of diastolic passive tension, the elastic sarcomeric protein titin, has been shown to be associated with HF, with unresolved involvement regarding its role at different heart rates. To determine whether titin is playing a role in the heart rate (frequency-) dependent acceleration of relaxation (FDAR). W, we studied the FDAR responses in live human left ventricular cardiomyocytes and the corresponding titin-based passive tension (TPT) from failing and non-failing human hearts., Methods and Results: Using atomic force, we developed a novel single-molecule force spectroscopy approach to detect TPT based on the frequency-modulated cardiac cycle. Mean TPT reduced upon an increased heart rate in non-failing human hearts, while this reduction was significantly blunted in failing human hearts. These mechanical changes in the titin distal Ig domain significantly correlated with the frequency-dependent relaxation kinetics of human cardiomyocytes obtained from the corresponding hearts. Furthermore, the data suggested that the higher the TPT, the faster the cardiomyocytes relaxed, but the lower the potential of myocytes to speed up relaxation at a higher heart rate. Such poorer FDAR response was also associated with a lesser reduction or a bigger increase in TPT upon elevated heart rate., Conclusions: Our study established a novel approach in detecting dynamic heart rate relevant tension changes physiologically on native titin domains. Using this approach, the data suggested that the regulation of kinetic reserve in cardiac relaxation and its pathological changes were associated with the intensity and dynamic changes of passive tension by titin., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been developed for cardiac cell transplantation studies more than a decade ago. In order to establish the hiPSC-CM-based platform as an autologous source for cardiac repair and drug toxicity, it is vital to understand the functionality of cardiomyocytes. Therefore, the goal of this study was to assess functional physiology, ultrastructural morphology, gene expression, and microRNA (miRNA) profiling at Wk-1, Wk-2 & Wk-4 in hiPSC-CMs in vitro. Functional assessment of hiPSC-CMs was determined by multielectrode array (MEA), Ca 2+ cycling and particle image velocimetry (PIV). Results demonstrated that Wk-4 cardiomyocytes showed enhanced synchronization and maturation as compared to Wk-1 & Wk-2. Furthermore, ultrastructural morphology of Wk-4 cardiomyocytes closely mimicked the non-failing (NF) adult human heart. Additionally, modulation of cardiac genes, cell cycle genes, and pluripotency markers were analyzed by real-time PCR and compared with NF human heart. Increasing expression of fatty acid oxidation enzymes at Wk-4 supported the switching to lipid metabolism. Differential regulation of 12 miRNAs was observed in Wk-1 vs Wk-4 cardiomyocytes. Overall, this study demonstrated that Wk-4 hiPSC-CMs showed improved functional, metabolic and ultrastructural maturation, which could play a crucial role in optimizing timing for cell transplantation studies and drug screening.

The force-frequency relationship (FFR) is an important regulatory mechanism that increases the force-generating capacity as well as the contraction and relaxation kinetics in human cardiac muscle as the heart rate increases. In human heart failure, the normally positive FFR often becomes flat, or even negative. The rate of cross-bridge cycling, which has been reported to affect cardiac output, could be potentially dysregulated and contribute to blunted or negative FFR in heart failure. We recently developed and herein use a novel method for measuring the rate of tension redevelopment. This method allows us to obtain an index of the rate of cross-bridge cycling in intact contracting cardiac trabeculae at physiological temperature and assess physiological properties of cardiac muscles while preserving posttranslational modifications representative of those that occur in vivo. We observed that trabeculae from failing human hearts indeed exhibit an impaired FFR and a reduced speed of relaxation kinetics. However, stimulation frequencies in the lower spectrum did not majorly affect cross-bridge cycling kinetics in nonfailing and failing trabeculae when assessed at maximal activation. Trabeculae from failing human hearts had slightly slower cross-bridge kinetics at 3 Hz as well as reduced capacity to generate force upon K + contracture at this frequency. We conclude that cross-bridge kinetics at maximal activation in the prevailing in vivo heart rates are not majorly impacted by frequency and are not majorly impacted by disease. NEW & NOTEWORTHY In this study, we confirm that cardiac relaxation kinetics are impaired in filing human myocardium and that cross-bridge cycling rate at resting heart rates does not contribute to this impaired relaxation. At high heart rates, failing myocardium cross-bridge rates are slower than in nonfailing myocardium.

Mineralocorticoid receptor (MR) drugs have been used clinically for decades to treat cardiovascular diseases. MR antagonists not only show preclinical efficacy for heart in Duchenne muscular dystrophy (DMD) models but also improve skeletal muscle force and muscle membrane integrity. The mechanisms of action of MR antagonists in skeletal muscles are entirely unknown. Since MR are present in many cell types in the muscle microenvironment, it is critical to define cell-intrinsic functions in each cell type to ultimately optimize antagonist efficacy for use in the widest variety of diseases. We generated a new conditional knockout of MR in myofibers and quantified cell-intrinsic mechanistic effects on functional and histological parameters in a DMD mouse model. Skeletal muscle MR deficiency led to improved respiratory muscle force generation and less deleterious fibrosis but did not reproduce MR antagonist efficacy on membrane susceptibility to induced damage. Surprisingly, acute application of MR antagonist to muscles led to improvements in membrane integrity after injury independent of myofiber MR. These data demonstrate that MR antagonists are efficacious to dystrophic skeletal muscles through both myofiber intrinsic effects on muscle force and downstream fibrosis and extrinsic functions on membrane stability. MR antagonists may therefore be applicable for treating more general muscle weakness and possibly other conditions that result from cell injuries., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Spectrins are cytoskeletal proteins essential for membrane biogenesis and regulation and serve critical roles in protein targeting and cellular signaling. αII spectrin ( SPTAN1 ) is one of two α spectrin genes and αII spectrin dysfunction is linked to alterations in axon initial segment formation, cortical lamination, and neuronal excitability. Furthermore, human αII spectrin loss-of-function variants cause neurological disease. As global αII spectrin knockout mice are embryonic lethal, the in vivo roles of αII spectrin in adult heart are unknown and untested. Here, based on pronounced alterations in αII spectrin regulation in human heart failure we tested the in vivo roles of αII spectrin in the vertebrate heart. We created a mouse model of cardiomyocyte-selective αII spectrin-deficiency (cKO) and used this model to define the roles of αII spectrin in cardiac function. αII spectrin cKO mice displayed significant structural, cellular, and electrical phenotypes that resulted in accelerated structural remodeling, fibrosis, arrhythmia, and mortality in response to stress. At the molecular level, we demonstrate that αII spectrin plays a nodal role for global cardiac spectrin regulation, as αII spectrin cKO hearts exhibited remodeling of αI spectrin and altered β-spectrin expression and localization. At the cellular level, αII spectrin deficiency resulted in altered expression, targeting, and regulation of cardiac ion channels Na V 1.5 and K V 4.3. In summary, our findings define critical and unexpected roles for the multifunctional αII spectrin protein in the heart. Furthermore, our work provides a new in vivo animal model to study the roles of αII spectrin in the cardiomyocyte., (© 2019 Lubbers et al.)

Introduction: When investigating drugs that treat heart diseases, it is critical when choosing an animal model for the said model to produce data that is translatable to the human patient population, while keeping in mind the principles of reduction, refinement, and replacement of the animal model in the research. Areas covered: In this review, the authors focus on mammalian models developed to study the impact of drug treatments on human heart failure. Furthermore, the authors address human patient variability and animal model invariability as well as the considerations that need to be made regarding choice of species. Finally, the authors discuss some of the most common models for the two most prominent human heart failure etiologies; increased load on the heart and myocardial ischemia. Expert opinion: In the authors' opinion, the data generated by drug studies is often heavily impacted by the choice of species and the physiologically relevant conditions under which the data are collected. Approaches that use multiple models and are not restricted to small rodents but involve some verification on larger mammals or on human myocardium, are needed to advance drug discovery for the very large patient population that suffers from heart failure.

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function and protects the heart from ischemia-reperfusion (I/R) injury. It is known that protein kinase-A (PKA)-mediated phosphorylation of cMyBP-C prevents I/R-dependent proteolysis, whereas dephosphorylation of cMyBP-C at PKA sites correlates with its degradation. While sites on cMyBP-C associated with phosphorylation and proteolysis co-localize, the mechanisms that link cMyBP-C phosphorylation and proteolysis during cardioprotection are not well understood. Therefore, we aimed to determine if abrogation of cMyBP-C proteolysis in association with calpain, a calcium-activated protease, confers cardioprotection during I/R injury. Calpain is activated in both human ischemic heart samples and ischemic mouse myocardium where cMyBP-C is dephosphorylated and undergoes proteolysis. Moreover, cMyBP-C is a substrate for calpain proteolysis and cleaved by calpain at residues 272-TSLAGAGRR-280, a domain termed as the calpain-target site (CTS). Cardiac-specific transgenic (Tg) mice in which the CTS motif was ablated were bred into a cMyBP-C null background. These Tg mice were conclusively shown to possess a normal basal structure and function by analysis of histology, electron microscopy, immunofluorescence microscopy, Q-space MRI of tissue architecture, echocardiography, and hemodynamics. However, the genetic ablation of the CTS motif conferred resistance to calpain-mediated proteolysis of cMyBP-C. Following I/R injury, the loss of the CTS reduced infarct size compared to non-transgenic controls. Collectively, these findings demonstrate the physiological significance of calpain-targeted cMyBP-C proteolysis and provide a rationale for studying inhibition of calpain-mediated proteolysis of cMyBP-C as a therapeutic target for cardioprotection., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background The aortic valve of the heart experiences constant mechanical stress under physiological conditions. Maladaptive valve injury responses contribute to the development of valvular heart disease. Here, we test the hypothesis that MG 53 (mitsugumin 53), an essential cell membrane repair protein, can protect valvular cells from injury and fibrocalcific remodeling processes associated with valvular heart disease. Methods and Results We found that MG 53 is expressed in pig and human patient aortic valves and observed aortic valve disease in aged Mg53-/- mice. Aortic valves of Mg53-/- mice showed compromised cell membrane integrity. In vitro studies demonstrated that recombinant human MG 53 protein protects primary valve interstitial cells from mechanical injury and that, in addition to mediating membrane repair, recombinant human MG 53 can enter valve interstitial cells and suppress transforming growth factor-β-dependent activation of fibrocalcific signaling. Conclusions Together, our data characterize valve interstitial cell membrane repair as a novel mechanism of protection against valvular remodeling and assess potential in vivo roles of MG 53 in preventing valvular heart disease.

Myocardial relaxation is critical for the heart to allow for adequate filling of the ventricles prior to the next contraction. In human heart failure, impairment of myocardial relaxation is a major problem, and impacts most patients suffering from end-stage failure. Furthering our understanding of myocardial relaxation is critical in developing future treatment strategies. This review highlights processes involved in myocardial relaxation, as well as governing processes that modulate myocardial relaxation, with a focus on impairment of myocardium-level relaxation in human end-stage heart failure., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aims: Heart failure (HF) is a prevalent disease that is considered the foremost reason for hospitalization in the United States. Most protein kinases (PK) are activated in heart disease and their inhibition has been shown to improve cardiac function in both animal and human studies. However, little is known about the direct impact of PKA and PKC inhibitors on human cardiac contractile function., Material and Methods: We investigated the ex vivo effect of such inhibitors on force as well as on kinetics of left ventricular (LV) trabeculae dissected from non-failing and failing human hearts. In these experiments, we applied 0.5 μM of H-89 and GF109203X, which are PKA and PKC inhibitors, respectively, in comparison to their vehicle DMSO (0.05%)., Key Findings and Conclusion: Statistical analyses revealed no significant effect for H-89 and GF109203X on either contractile force or kinetics parameters of both non-failing and failing muscles even though they were used at a concentration higher than the reported IC 50 s and K i s. Therefore, several factors such as selectivity, concentration, and treatment time, which are related to these PK inhibitors according to previous studies require further exploration., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In addition to their role in the central nervous system (CNS), N-methyl-d-aspartate (NMDA) receptors activation contributes to myocardial pathogenesis. This study sought to determine the potential cardioprotective effects of pre-treatment with memantine, an NMDA receptor antagonist, in heart failure (HF). A subcutaneous injection of isoproterenol (5 mg/kg/day) for 14 days was used for the induction of heart failure in rats. Memantine was injected intraperitoneally (ip) at doses of 5 and 20 mg/kg one week before isoproterenol injection for 21 days (n = 8 each group). Then, hemodynamic, electrocardiogram and histopathological changes as well as lipid peroxidation, myeloperoxidase (MPO) and adenosine monophosphate-activated protein kinase (AMPK) activity were evaluated. Histopathological analysis showed a marked attenuation of myocyte necrosis and fibrosis in memantine 20 mg/kg pre-treated group (p < 0.001) in comparison to HF group. Pre-treatment with memantine 20 mg/kg significantly reduced myocardial edematous, MPO activity and malondialdehyde (MDA) levels in comparison to HF group (p < 0.05, p < 0.05 and p < 0.001 respectively). Memantine had no significant effect on hemodynamic parameters and AMPK activity but improved the electrocardiogram (ECG) pattern. Our results for the first time showed cardioprotective effects of memantine in HF through reduction in cardiac remodeling, lipid peroxidation and neutrophil infiltration. In addition these effects are through an AMPK-independent pathways., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)