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Background: Myocardial infarction (MI) is a significant cause of death in diabetic patients. Growing evidence suggests that mitochondrial dysfunction contributes to heart failure in diabetes. However, the molecular mechanisms of mitochondrial dysfunction mediating heart failure in diabetes are still poorly understood., Methods: We examined MRPL12 levels in right atrial appendage tissues from diabetic patients undergoing coronary artery bypass graft (CABG) surgery. Using AC-16 cells overexpressing MRPL12 under normal and hyperglycemic conditions we performed mitochondrial functional assays OXPHOS, bioenergetics, mitochondrial membrane potential, ATP production and cell death., Results: We observed elevated MRPL12 levels in heart tissue samples from diabetic patients with ischemic heart disease compared to non-diabetic patients. Overexpression of MRPL12 under hyperglycemic conditions did not affect oxidative phosphorylation (OXPHOS) levels, cellular ATP levels, or cardiomyocyte cell death. However, notable impairment in mitochondrial membrane potential (MMP) was observed under hyperglycemic conditions, along with alterations in both basal respiration oxygen consumption rate (OCR) and maximal respiratory capacity OCR., Conclusions: Overall, our results suggest that MRPL12 may have a compensatory role in the diabetic myocardium with ischemic heart disease, suggesting that MRPL12 may implicate in the pathophysiology of MI in diabetes., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Background: A healthy heart is able to modify its function and increase relaxation through post-translational modifications of myofilament proteins. While there are known examples of serine/threonine kinases directly phosphorylating myofilament proteins to modify heart function, the roles of tyrosine (Y) phosphorylation to directly modify heart function have not been demonstrated. The myofilament protein TnI (troponin I) is the inhibitory subunit of the troponin complex and is a key regulator of cardiac contraction and relaxation. We previously demonstrated that TnI-Y26 phosphorylation decreases calcium-sensitive force development and accelerates calcium dissociation, suggesting a novel role for tyrosine kinase-mediated TnI-Y26 phosphorylation to regulate cardiac relaxation. Therefore, we hypothesize that increasing TnI-Y26 phosphorylation will increase cardiac relaxation in vivo and be beneficial during pathological diastolic dysfunction., Methods: The signaling pathway involved in TnI-Y26 phosphorylation was predicted in silico and validated by tyrosine kinase activation and inhibition in primary adult murine cardiomyocytes. To investigate how TnI-Y26 phosphorylation affects cardiac muscle, structure, and function in vivo, we developed a novel TnI-Y26 phosphorylation-mimetic mouse that was subjected to echocardiography, pressure-volume loop hemodynamics, and myofibril mechanical studies. TnI-Y26 phosphorylation-mimetic mice were further subjected to the nephrectomy/DOCA (deoxycorticosterone acetate) model of diastolic dysfunction to investigate the effects of increased TnI-Y26 phosphorylation in disease., Results: Src tyrosine kinase is sufficient to phosphorylate TnI-Y26 in cardiomyocytes. TnI-Y26 phosphorylation accelerates in vivo relaxation without detrimental structural or systolic impairment. In a mouse model of diastolic dysfunction, TnI-Y26 phosphorylation is beneficial and protects against the development of disease., Conclusions: We have demonstrated that tyrosine kinase phosphorylation of TnI is a novel mechanism to directly and beneficially accelerate myocardial relaxation in vivo., Competing Interests: Disclosures T.A. McKinsey is on the scientific advisory boards of Artemes Bio and Eikonizo Therapeutics, received funding from Italfarmaco for an unrelated project, and has a subcontract from Eikonizo Therapeutics for a small business innovation research grant from the National Institutes of Health (HL154959). The other authors report no conflicts.

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.)

For years, ejection fraction has been an essentially ubiquitous measurement for assessing the cardiovascular function of animal models in research labs. Despite technological advances, it remains the top choice among research labs for reporting heart function to this day, and is often overstated in applications. This unfortunately may lead to misinterpretation of data. Clinical approaches have now surpassed research methods, allowing for deeper analysis of the tiers of cardiovascular performance (cardiovascular performance, heart performance, systolic and diastolic function, and contractility). Analysis of each tier is crucial for understanding heart performance, mechanism of action, and disease diagnosis, classification, and progression. This review will elucidate the differences between the tiers of cardiovascular function and discuss the benefits of measuring each tier via speckle tracking echocardiography for basic scientists., 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 Sturgill, Shettigar and Ziolo.)

Fibroblasts produce the majority of collagen in the heart and are thought to regulate extracellular matrix (ECM) turnover. Although fibrosis accompanies many cardiac pathologies and is generally deleterious, the role of fibroblasts in maintaining the basal ECM network and in fibrosis in vivo is poorly understood. We genetically ablated fibroblasts in mice to evaluate the impact on homeostasis of adult ECM and cardiac function after injury. Fibroblast-ablated mice demonstrated a substantive reduction in cardiac fibroblasts, but fibrillar collagen and the ECM proteome were not overtly altered when evaluated by quantitative mass spectrometry and N-terminomics. However, the distribution and quantity of collagen VI, microfibrillar collagen that forms an open network with the basement membrane, was reduced. In fibroblast-ablated mice, cardiac function was better preserved following angiotensin II/phenylephrine (AngII/PE)-induced fibrosis and myocardial infarction (MI). Analysis of cardiomyocyte function demonstrated altered sarcomere shortening and slowed calcium decline in both uninjured and AngII/PE-infused fibroblast-ablated mice. After MI, the residual resident fibroblasts responded to injury, albeit with reduced proliferation and numbers immediately after injury. These results indicate that the adult mouse heart tolerates a significant degree of fibroblast loss with a potentially beneficial impact on cardiac function after injury. The cardioprotective effect of controlled fibroblast reduction may have therapeutic value in heart disease., Competing Interests: JK, AH, SB, GG, JC, KH, VS, AL, LD, JH, JJ, DT, MZ, SA, MT No competing interests declared, (© 2022, Kuwabara, Hara et al.)

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder causing progressive muscle degeneration. Although cardiomyopathy is a leading mortality cause in DMD patients, the mechanisms underlying heart failure are not well understood. Previously, we showed that NF-κB exacerbates DMD skeletal muscle pathology by promoting inflammation and impairing new muscle growth. Here, we show that NF-κB is activated in murine dystrophic (mdx) hearts, and that cardiomyocyte ablation of NF-κB rescues cardiac function. This physiological improvement is associated with a signature of upregulated calcium genes, coinciding with global enrichment of permissive H3K27 acetylation chromatin marks and depletion of the transcriptional repressors CCCTC-binding factor, SIN3 transcription regulator family member A, and histone deacetylase 1. In this respect, in DMD hearts, NF-κB acts differently from its established role as a transcriptional activator, instead promoting global changes in the chromatin landscape to regulate calcium genes and cardiac function.

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic syndrome characterized by sudden death. There are several genetic forms of CPVT associated with mutations in genes encoding the cardiac ryanodine receptor (RyR2) and its auxiliary proteins including calsequestrin (CASQ2) and calmodulin (CaM). It has been suggested that impairment of the ability of RyR2 to stay closed (ie, refractory) during diastole may be a common mechanism for these diseases. Here, we explore the possibility of engineering CaM variants that normalize abbreviated RyR2 refractoriness for subsequent viral-mediated delivery to alleviate arrhythmias in non-CaM-related CPVT., Methods and Results: To that end, we have designed a CaM protein (GSH-M37Q; dubbed as therapeutic CaM or T-CaM) that exhibited a slowed N-terminal Ca dissociation rate and prolonged RyR2 refractoriness in permeabilized myocytes derived from CPVT mice carrying the CASQ2 mutation R33Q. This T-CaM was introduced to the heart of R33Q mice through recombinant adeno-associated viral vector serotype 9. Eight weeks postinfection, we performed confocal microscopy to assess Ca handling and recorded surface ECGs to assess susceptibility to arrhythmias in vivo. During catecholamine stimulation with isoproterenol, T-CaM reduced isoproterenol-promoted diastolic Ca waves in isolated CPVT cardiomyocytes. Importantly, T-CaM exposure abolished ventricular tachycardia in CPVT mice challenged with catecholamines., Conclusions: Our results suggest that gene transfer of T-CaM by adeno-associated viral vector serotype 9 improves myocyte Ca handling and alleviates arrhythmias in a calsequestrin-associated CPVT model, thus supporting the potential of a CaM-based antiarrhythmic approach as a therapeutic avenue for genetically distinct forms of CPVT., (© 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

The contractile property of the myocardium is maintained by cell-cell junctions enabling cardiomyocytes to work as a syncytium. Alterations in cell-cell junctions are observed in heart failure, a disease characterized by the activation of Transforming Growth Factor beta 1 (TGFβ1). While TGFβ1 has been implicated in diverse biologic responses, its molecular function in controlling cell-cell adhesion in the heart has never been investigated. Cardiac-specific transgenic mice expressing active TGFβ1 were generated to model the observed increase in activity in the failing heart. Activation of TGFβ1 in the heart was sufficient to drive ventricular dysfunction. To begin to understand the function of this important molecule we undertook an extensive structural analysis of the myocardium by electron microscopy and immunostaining. This approach revealed that TGFβ1 alters intercalated disc structures and cell-cell adhesion in ventricular myocytes. Mechanistically, we found that TGFβ1 induces the expression of neural adhesion molecule 1 (NCAM1) in cardiomyocytes in a p38-dependent pathway, and that selective targeting of NCAM1 was sufficient to rescue the cell adhesion defect observed when cardiomyocytes were treated with TGFβ1. Importantly, NCAM1 was upregulated in human heart samples from ischemic and non-ischemic cardiomyopathy patients and NCAM1 protein levels correlated with the degree of TGFβ1 activity in the human cardiac ventricle. Overall, we found that TGFβ1 is deleterious to the heart by regulating the adhesion properties of cardiomyocytes in an NCAM1-dependent mechanism. Our results suggest that inhibiting NCAM1 would be cardioprotective, counteract the pathological action of TGFβ1 and reduce heart failure severity., (Published by Elsevier Ltd.)

The Ras homolog A (RhoA) subfamily of Rho guanosine triphosphatases (GTPases) regulates actin-based cellular functions in bone such as differentiation, migration, and mechanotransduction. Polymorphisms or genetic ablation of RHOA and some of its regulatory guanine exchange factors (GEFs) have been linked to poor bone health in humans and mice, but the effects of RhoA-specific GTPase-activating proteins (GAPs) on bone quality have not yet been identified. Therefore, we examined the consequences of RhoGAP Myo9b gene knockout on bone growth, phenotype, and cellular activity. Male and female mice lacking both alleles demonstrated growth retardation and decreased bone formation rates during early puberty. These mice had smaller, weaker bones by 4 weeks of age, but only female KOs had altered cellular numbers, with fewer osteoblasts and more osteoclasts. By 12 weeks of age, bone quality in KOs worsened. In contrast, 4-week-old heterozygotes demonstrated bone defects that resolved by 12 weeks of age. Throughout, Myo9b ablation affected females more than males. Osteoclast activity appeared unaffected. In primary osteogenic cells, Myo9b was distributed in stress fibers and focal adhesions, and its absence resulted in poor spreading and eventual detachment from culture dishes. Similarly, MC3T3-E1 preosteoblasts with transiently suppressed Myo9b levels spread poorly and contained decreased numbers of focal adhesions. These cells also demonstrated reduced ability to undergo IGF-1-induced spreading or chemotaxis toward IGF-1, though responses to PDGF and BMP-2 were unaffected. IGF-1 receptor (IGF1R) activation was normal in cells with diminished Myo9b levels, but the activated receptor was redistributed from stress fibers and focal adhesions into nuclei, potentially affecting receptor accessibility and gene expression. These results demonstrate that Myo9b regulates a subset of RhoA-activated processes necessary for IGF-1 responsiveness in osteogenic cells, and is critical for normal bone formation in growing mice. © 2017 American Society for Bone and Mineral Research., (© 2017 American Society for Bone and Mineral Research.)

Background: Particulate matter (PM; PM 2.5 [PM with diameters of <2.5 μm]) exposure during development is strongly associated with adverse cardiovascular outcomes at adulthood. In the present study, we tested the hypothesis that in utero PM 2.5 exposure alone could alter cardiac structure and function at adulthood., Methods and Results: Female FVB mice were exposed either to filtered air or PM 2.5 at an average concentration of 73.61 μg/m 3 for 6 h/day, 7 days/week throughout pregnancy. After birth, animals were analyzed at 12 weeks of age. Echocardiographic (n=9-10 mice/group) and pressure-volume loop analyses (n=5 mice/group) revealed reduced fractional shortening, increased left ventricular end-systolic and -diastolic diameters, reduced left ventricular posterior wall thickness, end-systolic elastance, contractile reserve (dP/dt max /end-systolic volume), frequency-dependent acceleration of relaxation), and blunted contractile response to β-adrenergic stimulation in PM 2.5 -exposed mice. Isolated cardiomyocyte (n=4-5 mice/group) function illustrated reduced peak shortening, ±dL/dT, and prolonged action potential duration at 90% repolarization. Histological left ventricular analyses (n=3 mice/group) showed increased collagen deposition in in utero PM 2.5 -exposed mice at adulthood. Cardiac interleukin (IL)-6, IL-1ß, collagen-1, matrix metalloproteinase (MMP) 9, and MMP13 gene expressions were increased at birth in in utero PM 2.5 -exposed mice (n=4 mice/group). In adult hearts (n=5 mice/group), gene expressions of sirtuin (Sirt) 1 and Sirt2 were decreased, DNA methyltransferase (Dnmt) 1, Dnmt3a, and Dnmt3b were increased, and protein expression (n=6 mice/group) of Ca 2+ -ATPase, phosphorylated phospholamban, and Na + /Ca 2+ exchanger were decreased., Conclusions: In utero PM 2.5 exposure triggers an acute inflammatory response, chronic matrix remodeling, and alterations in Ca 2+ handling proteins, resulting in global adult cardiac dysfunction. These results also highlight the potential involvement of epigenetics in priming of adult cardiac disease., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Plants commonly respond to stressors by modulating the expression of a large family of calcium binding proteins including isoforms of the ubiquitous signaling protein calmodulin (CaM). The various plant CaM isoforms are thought to differentially regulate the activity of specific target proteins to modulate cellular stress responses. The mechanism(s) behind differential target activation by the plant CaMs is unknown. In this study, we used steady-state and stopped-flow fluorescence spectroscopy to investigate the strategy by which two soybean CaMs (sCaM1 and sCaM4) have evolved to differentially regulate NAD kinase (NADK), which is activated by sCaM1 but inhibited by sCaM4. Although the isolated proteins have different cation binding properties, in the presence of Mg 2+ and the CaM binding domains from proteins that are differentially regulated, the two plant CaMs respond nearly identically to rapid and slow Ca 2+ transients. Our data suggest that the plant CaMs have evolved to bind certain targets with comparable affinities, respond similarly to a particular Ca 2+ signature, but achieve different structural states, only one of which can activate the enzyme. Understanding the basis for differential enzyme regulation by the plant CaMs is the first step to engineering a vertebrate CaM that will selectively alter the CaM signaling network.

Throughout history, muscle research has led to numerous scientific breakthroughs that have brought insight to a more general understanding of all biological processes. Potentially one of the most influential discoveries was the role of the second messenger calcium and its myriad of handling and sensing systems that mechanistically control muscle contraction. In this review we will briefly discuss the significance of calcium as a universal second messenger along with some of the most common calcium binding motifs in proteins, focusing on the EF-hand. We will also describe some of our approaches to rationally design calcium binding proteins to palliate, or potentially even cure cardiovascular disease. Considering not all failing hearts have the same etiology, genetic background and co-morbidities, personalized therapies will need to be developed. We predict designer proteins will open doors for unprecedented personalized, and potentially, even generalized medicines as gene therapy or protein delivery techniques come to fruition., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Treatment for heart disease, the leading cause of death in the world, has progressed little for several decades. Here we develop a protein engineering approach to directly tune in vivo cardiac contractility by tailoring the ability of the heart to respond to the Ca(2+) signal. Promisingly, our smartly formulated Ca(2+)-sensitizing TnC (L48Q) enhances heart function without any adverse effects that are commonly observed with positive inotropes. In a myocardial infarction (MI) model of heart failure, expression of TnC L48Q before the MI preserves cardiac function and performance. Moreover, expression of TnC L48Q after the MI therapeutically enhances cardiac function and performance, without compromising survival. We demonstrate engineering TnC can specifically and precisely modulate cardiac contractility that when combined with gene therapy can be employed as a therapeutic strategy for heart disease.

In the mol-ecules of the title compounds, (2E)-1-(3-bromo-thio-phen-2-yl)-3-(2-meth-oxy-phen-yl)prop-2-en-1-one, C14H11BrO2S, (I), which crystallizes in the space group P-1 with four independent mol-ecules in the asymmetric unit (Z' = 8), and (2E)-1-(3-bromo-thio-phen-2-yl)-3-(3,4-di-meth-oxy-phen-yl)prop-2-en-1-one, C15H13BrO3S, (II), which crystallizes with Z' = 8 in the space group I2/a, the non-H atoms are nearly coplanar. The mol-ecules of (I) pack with inversion symmetry stacked diagonally along the a-axis direction. Weak C-H⋯Br intra-molecular inter-actions in each of the four mol-ecules in the asymmetric unit are observed. In (II), weak C-H⋯O, bifurcated three-center inter-molecular inter-actions forming dimers along with weak C-H⋯π and π-π stacking inter-actions are observed, linking the mol-ecules into sheets along [001]. A weak C-H⋯Br intra-molecular inter-action is also present. There are no classical hydrogen bonds present in either structure.

Parvalbumin (PV), an EF-hand protein family member, is a delayed calcium buffer that exchanges magnesium for calcium to facilitate fast skeletal muscle relaxation. Genetic approaches that express parvalbumin in the heart also enhance relaxation and show promise of being therapeutic against various cardiac diseases where relaxation is compromised. Unfortunately, skeletal muscle PVs have very slow rates of Ca(2+) dissociation and are prone to becoming saturated with Ca(2+), eventually losing their buffering capability within the constantly beating heart. In order for PV to have a more therapeutic potential in the heart, a PV with faster rates of calcium dissociation and high Mg(2+) affinity is needed. We demonstrate that at 35°C, rat β-PV has an ~30-fold faster rate of Ca(2+) dissociation compared to rat skeletal muscle α-PV, and still possesses a physiologically relevant Ca(2+) affinity (~100 nM). However, rat β-PV will not be a delayed Ca(2+) buffer since its Mg(2+) affinity is too low (~1 mM). We have engineered two mutations into rat β-PV, S55D and E62D, when observed alone increase Mg(2+) affinity up to fivefold, but when combined increase Mg(2+) affinity ~13-fold, well within a physiologically relevant affinity. Furthermore, the Mg(2+) dissociation rate (172/s) from the engineered S55D, E62D PV is slow enough for delayed Ca(2+) buffering. Additionally, the engineered PV retains a high Ca(2+) affinity (132 nM) and fast rate of Ca(2+) dissociation (64/s). These PV design strategies hold promise for the development of new therapies to remediate relaxation abnormalities in different heart diseases and heart failure.