Roles of cMyBP-C phosphorylation on cardiac contractile dysfunction in db/db mice.

Type 2 diabetes mellitus (T2DM) is a metabolic disease and comorbidity associated with several conditions, including cardiac dysfunction leading to heart failure with preserved ejection fraction (HFpEF), in turn resulting in T2DM-induced cardiomyopathy (T2DM-CM). However, the molecular mechanisms underlying the development of T2DM-CM are poorly understood. It is hypothesized that molecular alterations in myopathic genes induced by diabetes promote the development of HFpEF, whereas cardiac myosin inhibitors can rescue the resultant T2DM-mediated cardiomyopathy. To test this hypothesis, a Leptin receptor-deficient db/db homozygous (Lepr db/db) mouse model was used to define the pathogenesis of T2DM-CM. Echocardiographic studies at 4 and 6 months revealed that Lepr db/db hearts started developing cardiac dysfunction by four months, and left ventricular hypertrophy with diastolic dysfunction was evident at 6 months. RNA-seq data analysis, followed by functional enrichment, revealed the differential regulation of genes related to cardiac dysfunction in Lepr db/db heart tissues. Strikingly, the level of cardiac myosin binding protein-C phosphorylation was significantly increased in Lepr db/db mouse hearts. Finally, using isolated skinned papillary muscles and freshly isolated cardiomyocytes, CAMZYOS ® (mavacamten, MYK-461), a prescription heart medicine used for symptomatic obstructive hypertrophic cardiomyopathy treatment, was tested for its ability to rescue T2DM-CM. Compared with controls, MYK-461 significantly reduced force generation in papillary muscle fibers and cardiomyocyte contractility in the db/db group. This line of evidence shows that 1) T2DM-CM is associated with hyperphosphorylation of cardiac myosin binding protein-C and 2) MYK-461 significantly lessened disease progression in vitro, suggesting its promise as a treatment for HFpEF.

MYBPC3 D389V Variant Induces Hypercontractility in Cardiac Organoids.

BACKGROUND: MYBPC3 , encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about the underlying etiology, additional in vitro studies are crucial to defining the underlying molecular mechanisms. Accordingly, this study aimed to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with a polymorphic variant (D389V) in MYBPC3 by using human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs). METHODS: The hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3 D389V variant. This variant is associated with increased fractional shortening and is highly prevalent in South Asian descendants. Recombinant C0-C2, N'-region of cMyBP-C (wildtype and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays in vitro . RESULTS: Confocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3 D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility with increased contraction velocity, faster calcium cycling, and faster contractile kinetics in hCOs expressing MYBPC3 D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3 D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential (ΔΨm). Next, spatial mapping revealed the presence of endothelial cells, fibroblasts, macrophages, immune cells, and cardiomyocytes in the hCOs. The hypercontractile function was significantly improved after treatment with the myosin inhibitor mavacamten (CAMZYOS®) in MYBPC3 D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3 D389V with myosin S2 region as a likely mechanism for hypercontraction. CONCLUSIONS: Conceptually, we showed the feasibility of assessing the functional and molecular mechanisms of HCM using highly translatable hCOs through pragmatic experiments that led to determining the MYBPC3 D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and Significance: What Is Known?: MYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. D389V is a polymorphic variant of MYBPC3 predicted to be present in 53000 US South Asians owing to the founder effect. D389V carriers have shown evidence of hyperdynamic heart, and human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes with D389V show cellular hypertrophy and irregular calcium transients. The molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined.What New Information Does This Article Contribute ?: The authors leveraged a highly translational cardiac organoid model to explore the role of altered cardiac calcium handling and cardiac contractility as a common pathway leading to pathophysiological phenotypes in patients with early HCM. The MYBPC3 D389V -mediated pathological pathway is first studied here by comparing functional properties using three-dimensional cardiac organoids differentiated from hiPSC and determining the presence of hypercontraction. Our data demonstrate that faster sarcomere kinetics resulting from lower binding affinity between D389V-mutated cMyBP-C protein and myosin S2, as evidenced by in vitro studies, could cause hypercontractility which was rescued by administration of mavacamten (CAMZYOS®), a myosin inhibitor. In addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential (ΔΨm), highlighting a possible early adaptive response to primary sarcomeric changes. Early treatment of MYBPC3 D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. GRAPHICAL ABSTRACT: A graphical abstract is available for this article.

Evaluate the in vitro effect of anthracycline and alkylating cytophosphane chemotherapeutics on dopaminergic neurons.

BACKGROUND: Iatrogenesis is an inevitable global threat to healthcare that drastically increases morbidity and mortality. Cancer is a fatal pathological condition that affects people of different ages, sexes, and races around the world. In addition to the detrimental cancer pathology, one of the most common contraindications and challenges observed in cancer patients is severe adverse drug effects and hypersensitivity reactions induced by chemotherapy. Chemotherapy-induced cognitive neurotoxicity is clinically referred to as Chemotherapy-induced cognitive impairment (CICI), chemobrain, or chemofog. In addition to CICI, chemotherapy also causes neuropsychiatric issues, mental disorders, hyperarousal states, and movement disorders. A synergistic chemotherapy regimen of Doxorubicin (Anthracycline-DOX) and Cyclophosphamide (Alkylating Cytophosphane-CPS) is indicated for the management of various cancers (breast cancer, lymphoma, and leukemia). Nevertheless, there are limited research studies on Doxorubicin and Cyclophosphamide's pharmacodynamic and toxicological effects on dopaminergic neuronal function. AIM: This study evaluated the dopaminergic neurotoxic effects of Doxorubicin and Cyclophosphamide. METHODS AND RESULTS: Doxorubicin and Cyclophosphamide were incubated with dopaminergic (N27) neurons. Neuronal viability was assessed using an MTT assay. The effect of Doxorubicin and Cyclophosphamide on various prooxidants, antioxidants, mitochondrial Complex-I & IV activities, and BAX expression were evaluated by Spectroscopic, Fluorometric, and RT-PCR methods, respectively. Prism-V software (La Jolla, CA, USA) was used for statistical analysis. Chemotherapeutics dose-dependently inhibited the proliferation of the dopaminergic neurons. The dopaminergic neurotoxic mechanism of Doxorubicin and Cyclophosphamide was attributed to a significant increase in prooxidants, a decrease in antioxidants, and augmented apoptosis without affecting mitochondrial function. CONCLUSION: This is one of the first reports that reveal Doxorubicin and Cyclophosphamide induce significant dopaminergic neurotoxicity. Thus, Chemotherapy-induced adverse drug reaction issues substantially persist during and after treatment and sometimes never be completely resolved clinically. Consequently, failure to adopt adequate patient care measures for cancer patients treated with certain chemotherapeutics might substantially raise the incidence of numerous movement disorders.

Heterogeneous Distribution of Genetic Mutations in Myosin Binding Protein-C Paralogs.

Myosin binding protein-C (MyBP-C) is a sarcomeric protein which regulates the force of contraction in striated muscles. Mutations in the MYBPC family of genes, including slow skeletal (MYBPC1), fast skeletal (MYBPC2) and cardiac (MYBPC3), can result in cardiac and skeletal myopathies. Nonetheless, their evolutionary pattern, pathogenicity and impact on MyBP-C protein structure remain to be elucidated. Therefore, the present study aimed to systematically assess the evolutionarily conserved and epigenetic patterns of MYBPC family mutations. Leveraging a machine learning (ML) approach, the Genome Aggregation Database (gnomAD) provided variants in MYBPC1, MYBPC2, and MYBPC3 genes. This was followed by an analysis with Ensembl's variant effect predictor (VEP), resulting in the identification of 8,618, 3,871, and 3,071 variants in MYBPC1, MYBPC2, and MYBPC3, respectively. Missense variants comprised 61%-66% of total variants in which the third nucleotide positions in the codons were highly altered. Arginine was the most mutated amino acid, important because most disease-causing mutations in MyBP-C proteins are arginine in origin. Domains C5 and C6 of MyBP-C were found to be hotspots for most mutations in the MyBP-C family of proteins. A high percentage of truncated mutations in cMyBP-C cause cardiomyopathies. Arginine and glutamate were the top hits in fMyBP-C and cMyBP-C, respectively, and tryptophan and tyrosine were the most common among the three paralogs changing to premature stop codons and causing protein truncations at the carboxyl terminus. A heterogeneous epigenetic pattern was identified among the three MYBP-C paralogs. Overall, it was shown that databases using computational approaches can facilitate diagnosis and drug discovery to treat muscle disorders caused by MYBPC mutations.

A clinically relevant combination treatment with doxorubicin and cyclophosphamide does not induce hepatotoxicity in C57BL/6J mice.

BACKGROUND AND AIM: Chronic exposure to chemotherapeutics can lead to severe adverse events including hepatotoxicity. A combination chemotherapy regimen of doxorubicin (DOX) and cyclophosphamide (CPS) is employed in treatment of several cancers such as leukemia, lymphoma, and breast cancer. It is not well understood whether a combination therapy of DOX and CPS can induce hepatotoxicity. We therefore sought to determine whether co-administration of DOX and CPS at their clinically relevant doses and frequency results in hepatotoxicity. METHODS: Male C57BL/6J mice received one intraperitoneal injection of saline or DOX-2mg /kg and CPS-50mg/kg once a week for 4 weeks. After the treatment period, liver histology and various serum biomarkers of hepatotoxicity were assessed. RESULTS: Co-treatment of DOX and CPS did not alter the serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin, albumin, globulin, or total protein. Similarly, co-administration of DOX and CPS did not result in a noticeable change in liver histology. However, it was notable that the concomitant treatment with DOX and CPS resulted in a significant increase in serum levels of aspartate aminotransferase (AST). Elevated serum AST levels were also associated with increased serum creatinine kinase (CK) levels, suggesting that the elevated serum AST levels are likely due to muscle injury following the co-administration of DOX and CPS. CONCLUSION: Taken together, our results, for the first time, suggest that co-administration of DOX and CPS, at their clinically relevant doses and frequency does not induce a significant hepatotoxicity in the mice.

Oroxylum indicum extract, at a physiologically relevant dosage, does not induce hepatotoxicity in C57BL/6J mice.

BACKGROUND: Botanical supplements have been proven to provide beneficial health effects. However, they can induce unintended adverse events such as hepatotoxicity. Oroxylum indicum extract (OIE, Sabroxy®) has several health benefits including anti-inflammatory, anti-arthritic, antifungal, antibacterial, and neuroprotective effects. It is currently unknown whether OIE has the potential to induce hepatotoxicity. PURPOSE: In the current study, we sought to determine whether OIE can induce hepatotoxicity in C57BL/6J mouse model. METHODS: The male mice were fed powdered rodent food (control group) or powdered rodent food mixed with OIE (Sabroxy®, 500mg/kg) daily for 4 weeks. Following the treatment, we assessed liver histology and serum levels of biomarkers commonly associated with liver damage, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). RESULTS: No significant alterations were observed in liver histology, and serum levels of ALT, AST, ALP, bilirubin, albumin, globulin and total protein in the OIE fed mice compared to the control mice. CONCLUSION: Taken together, our results suggest that OIE, when fed at its physiologically relevant dosage, does not induce hepatotoxicity in C57BL/6J mice.

Oroxylum Indicum ameliorates chemotherapy induced cognitive impairment.

While chemotherapy is the most effective therapeutic approach for treating a variety of cancer patients, commonly used chemotherapeutic agents, often induce several adverse effects. Escalating evidence indicates that chemotherapeutics, particularly doxorubicin (DOX) and cyclophosphamide (CPS), induce cognitive impairment associated with central nervous system toxicity. This study was performed to determine neuroprotective effects of Oroxylum indicum extract (OIE) in regard to preventing chemotherapy induced cognitive impairment (CICI) occurring after 4 cycles of DOX (2mg/kg) and CPS (50mg/kg) combination chemotherapy in male C57BL/6J mice. OIE significantly prevented the chemotherapy impaired short-term cognitive performance, exploratory behavior associated with cognitive performance, cognitive performance, and spatial learning and memory in the Y-maze, Open-Field, Novel Object Recognition, and Morris Water Maze tests, respectively. These data suggest that OIE protects from the CICI. OIE decreased the reactive oxygen species and lipid peroxide generated by the chemotherapy treatment in the brain, while also blocking the chemotherapy-induced glutathione depletion. These results establish that OIE exhibits potent antioxidant activity in chemotherapy treated mice. Notably, OIE significantly increased the Complex-I and Complex-IV activities in the brain, indicating that OIE enhances mitochondrial function in the brain. In silico analysis of the major active chemical constituents (Oroxylin A, Baicalein and Chrysin) of OIE indicated that OIE has a favorable absorption, distribution, metabolism and excretion (ADME) profile. Taken together, our results are consistent with the conclusion that OIE prevents CICI by counteracting oxidative stress and perhaps by improving mitochondrial function.

Genetic, clinical, molecular, and pathogenic aspects of the South Asian-specific polymorphic MYBPC3Δ25bp variant.

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by ventricular enlargement, diastolic dysfunction, and increased risk for sudden cardiac death. Sarcomeric genetic defects are the predominant known cause of HCM. In particular, mutations in the myosin-binding protein C gene (MYBPC3) are associated with ~ 40% of all HCM cases in which a genetic basis has been established. A decade ago, our group reported a 25-base pair deletion in intron 32 of MYBPC3 (MYBPC3Δ25bp) that is uniquely prevalent in South Asians and is associated with autosomal dominant cardiomyopathy. Although our studies suggest that this deletion results in left ventricular dysfunction, cardiomyopathies, and heart failure, the precise mechanism by which this variant predisposes to heart disease remains unclear. Increasingly appreciated, however, is the contribution of secondary risk factors, additional mutations, and lifestyle choices in augmenting or modifying the HCM phenotype in MYBPC3Δ25bp carriers. Therefore, the goal of this review article is to summarize the current research dedicated to understanding the molecular pathophysiology of HCM in South Asians with the MYBPC3Δ25bp variant. An emphasis is to review the latest techniques currently applied to explore the MYBPC3Δ25bp pathogenesis and to provide a foundation for developing new diagnostic strategies and advances in therapeutics.

Distal Arthrogryposis and Lethal Congenital Contracture Syndrome - An Overview.

Distal arthrogryposis (DA) is a skeletal muscle disorder which can be classified under a broader term as Arthrogryposis multiplex contractures. DA is characterized by the presence of joint contractures at various parts of the body, particularly in distal extremities. It is identified as an autosomal dominant and a rare X-linked recessive disorder associated with increased connective tissue formation around joints in such way that immobilizes muscle movement causing deformities. DA is again classified into various types since it manifests as a range of conditions representing different etiologies. Myopathy is one of the most commonly listed etiologies of DA. The mutations in sarcomeric protein-encoding genes lead to decreased sarcomere integrity, which is often associated with this disorder. Also, skeletal disorders are often associated with cardiac disorders. Some studies mention the presence of cardiomyopathy in patients with skeletal dysfunction. Therefore, it is hypothesized that the congenitally mutated protein that causes DA can also lead to cardiomyopathy. In this review, we will summarize the different forms of DA and their clinical features, along with gene mutations responsible for causing DA in its different forms. We will also examine reports that list mutations also known to cause heart disorders in the presence of DA.

Predictable hematological markers associated with cognitive decline in valid rodent models of cognitive impairment.

Endogenous (hyperglycemia) and exogenous (therapeutic, prophylactic, street drugs) factors can considerably contribute to cognitive impairment (CI). Currently, there are few invasive and/or noninvasive markers that correlate with CI and those that do exist require expensive or invasive techniques to predict and accurately measure the cognitive decline. Therefore, we sought to determine hematological markers as predictors of CI in two different chemically induced valid rodent models of CI (streptozotocin induced hyperglycemic model and chemotherapy [doxorubicin/cyclophosphamide] treated rodent model). Hematological markers were analyzed in the above rodent models of CI CI and compared to their respective control groups. There was a significant increase in creatinine kinase, lactate dehydrogenase and aspartate aminotransferase (AST) in the chemotherapy group. Blood urea nitrogen (BUN), alkaline phosphatase (ALP), bilirubin, creatinine and glucose levels were significantly increased in the streptozotocin group. Interestingly, triglycerides were significantly elevated in both the streptozotocin and chemotherapy groups. Previous studies with human subjects have shown a potential link between the increase in triglyceride levels and CI. Likewise, our data indicate a notable correlation with an increase in triglycerides to cognitive impairment in the rodent models. This suggests elevated levels of triglycerides could prove to be a potential noninvasive hematological marker for the increased risk of CI. Further studies are warranted to determine the causal relationship between elevated triglyceride levels and CI.