Guidelines on antibody use in physiology research.

Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research.

Aging Reveals a Sex-Dependent Susceptibility of Sarcospan-Deficient Mice to Cardiometabolic Disease.

Numerous genes including sarcospan (SSPN) have been designated as obesity-susceptibility genes by human genome-wide association studies. Variants in the SSPN locus have been linked with sex-dependent obesity-associated traits, however this association has not been investigated in vivo. To delineate the role SSPN plays in regulating metabolism with potential to impact cardiac function we subjected young and aged global SSPN-deficient (SSPN-/-) male and female mice to obesogenic conditions (60% fat diet). We hypothesized that loss of SSPN combined with metabolic stress would increase susceptibility of mice to cardiometabolic disease. Baseline and endpoint assessments of several anthropometric parameters were performed including weight, glucose tolerance, and fat distribution of mice fed control (CD) and high fat diet (HFD). Doppler echocardiography was used to monitor cardiac function. White adipose and cardiac tissues were assessed for inflammation utilizing histological, gene expression and cytokine analysis. Overall, SSPN deficiency protected both sexes and ages from diet-induced obesity with a greater effect in females. While SSPN-/- HFD mice gained less weight than WT cohorts, SSPN-/- CD groups increased weight. Furthermore, aged SSPN-/- mice developed glucose intolerance regardless of diet. Echocardiography showed preserved systolic function for all groups, however aged SSPN-/- males (CD) exhibited significant increases in LVmass and (HFD) signs of diastolic dysfunction. Cytokine analysis revealed significantly increased IL-1α and IL-17A in white adipose tissue from young SSPN-/- male mice that may be protective from diet-induced obesity. Overall, these studies suggest several sex-dependent mechanisms influence the role SSPN plays in metabolic responses that become evident with age.

Sarcospan Deficiency Increases Oxidative Stress and Arrhythmias in Hearts after Acute Ischemia-Reperfusion Injury.

The protein sarcospan (SSPN) is an integral member of the dystrophin-glycoprotein complex (DGC) and has been shown to be important in the heart during the development and the response to acute stress. In this study, we investigated the role of SSPN in the cardiac response to acute ischemia-reperfusion (IR) injury in SSPN-deficient (SSPN-/-) mice. First, the hemodynamic response of SSPN-/- mice was tested and was similar to SSPN+/+ (wild-type) mice after isoproterenol injection. Using the in situ Langendorff perfusion method, SSPN-/- hearts were subjected to IR injury and found to have increased infarct size and arrhythmia susceptibility compared to SSPN+/+. Ca2+ handling was assessed in single cardiomyocytes and diastolic Ca2+ levels were increased after acute ß-AR stimulation in SSPN+/+ but not SSPN-/-. It was also found that SSPN-/- cardiomyocytes had reduced Ca2+ SR content compared to SSPN+/+ but similar SR Ca2+ release. Next, we used qRT-PCR to examine gene expression of Ca2+ handling proteins after acute IR injury. SSPN-/- hearts showed a significant decrease in L-type Ca2+ channels and a significant increase in Ca2+ release channel (RyR2) expression. Interestingly, under oxidizing conditions reminiscent of IR, SSPN-/- cardiomyocytes, had increased H2O2-induced reactive oxygen species production compared to SSPN+/+. Examination of oxidative stress proteins indicated that NADPH oxidase 4 and oxidized CAMKII were increased in SSPN-/- hearts after acute IR injury. These results suggest that increased arrhythmia susceptibility in SSPN-/- hearts post-IR injury may arise from alterations in Ca2+ handling and a reduced capacity to regulate oxidative stress pathways.

On 'The content of troponin, tropomyosin, actin, and myosin in rabbit skeletal muscle myofibrils' by James D. Potter.

After the discovery of troponin by Ebashi almost sixty years ago the field of striated muscle regulation has made significant progress. In the 1970's the nascent troponin field gained momentum, including contributions by James D. Potter who established the stoichiometry of contractile proteins in the myofibril (Arch Biochem Biophys. 1974 Jun; 162(2):436-41. https://doi.org/10.1016/0003-9861(7490202-1)). This opened the door to refinement of competing models that described possible thick filament configurations. This study suggested the presence of one myosin per cross bridge and provided accurate calculations of the molar ratios of each protein - myosin: actin: tropomyosin: troponin T: troponin I: troponin C.

A comprehensive guide to genetic variants and post-translational modifications of cardiac troponin C.

Familial cardiomyopathy is an inherited disease that affects the structure and function of heart muscle and has an extreme range of phenotypes. Among the millions of affected individuals, patients with hypertrophic (HCM), dilated (DCM), or left ventricular non-compaction (LVNC) cardiomyopathy can experience morphologic changes of the heart which lead to sudden death in the most detrimental cases. TNNC1, the gene that codes for cardiac troponin C (cTnC), is a sarcomere gene associated with cardiomyopathies in which probands exhibit young age of presentation and high death, transplant or ventricular fibrillation events relative to TNNT2 and TNNI3 probands. Using GnomAD, ClinVar, UniProt and PhosphoSitePlus databases and published literature, an extensive list to date of identified genetic variants in TNNC1 and post-translational modifications (PTMs) in cTnC was compiled. Additionally, a recent cryo-EM structure of the cardiac thin filament regulatory unit was used to localize each functionally studied amino acid variant and each PTM (acetylation, glycation, s-nitrosylation, phosphorylation) in the structure of cTnC. TNNC1 has a large number of variants (> 100) relative to other genes of the same transcript size. Surprisingly, the mapped variant amino acids and PTMs are distributed throughout the cTnC structure. While many cardiomyopathy-associated variants are localized in α-helical regions of cTnC, this was not statistically significant χ2 (p = 0.72). Exploring the variants in TNNC1 and PTMs of cTnC in the contexts of cardiomyopathy association, physiological modulation and potential non-canonical roles provides insights into the normal function of cTnC along with the many facets of TNNC1 as a cardiomyopathic gene.

Meta-analysis of cardiomyopathy-associated variants in troponin genes identifies loci and intragenic hot spots that are associated with worse clinical outcomes.

INTRODUCTION: Troponin (TNN)-encoded cardiac troponins (Tn) are critical for sensing calcium and triggering myofilament contraction. TNN variants are associated with development of cardiomyopathy; however, recent advances in genetic analysis have identified rare population variants. It is unclear how certain variants are associated with disease while others are tolerated. OBJECTIVE: To compare probands with TNNT2, TNNI3, and TNNC1 variants and utilize high-resolution variant comparison mapping of pathologic and rare population variants to identify loci associated with disease pathogenesis. METHODS: Cardiomyopathy-associated TNN variants were identified in the literature and topology mapping conducted. Clinical features were compiled and compared. Rare population variants were obtained from the gnomAD database. Signal-to-noise (S:N) normalized pathologic variant frequency against population variant frequency. Abstract review of clinical phenotypes was applied to "significant" hot spots. RESULTS: Probands were compiled (N = 70 studies, 224 probands) as were rare variants (N = 125,748 exomes; 15,708 genomes, MAF <0.001). TNNC1-positive probands demonstrated the youngest age of presentation (20.0 years; P = .016 vs TNNT2; P = .004 vs TNNI3) and the highest death, transplant, or ventricular fibrillation events (P = .093 vs TNNT2; P = .024 vs TNNI3; Kaplan Meir: P = .025). S:N analysis yielded hot spots of diagnostic significance within the tropomyosin-binding domains, α-helix 1, and the N-Terminus in TNNT2 with increased sudden cardiac death and ventricular fibrillation (P = .004). The inhibitory region and C-terminal region in TNNI3 exhibited increased restrictive cardiomyopathy (P =.008). HCM and RCM models tended to have increased calcium sensitivity and DCM decreased sensitivity (P < .001). DCM and HCM studies typically showed no differences in Hill coefficient which was decreased in RCM models (P < .001). CM models typically demonstrated no changes to Fmax (P = .239). CONCLUSION: TNNC1-positive probands had younger ages of diagnosis and poorer clinical outcomes. Mapping of TNN variants identified locations in TNNT2 and TNNI3 associated with heightened pathogenicity, RCM diagnosis, and increased risk of sudden death.

The missing links within troponin.

The cardiac contraction-relaxation cycle is controlled by a sophisticated set of machinery. Of particular interest, is the revelation that allosteric networks transmit effects of binding at one site to influence troponin complex dynamics and structural-mediated signaling in often distal, functional sites in the myofilament. Our recent observations provide compelling evidence that allostery can explain the function of large-scale macromolecular events. Here we elaborate on our recent findings of interdomain communication within troponin C, using cutting-edge structural biology approaches, and highlight the importance of unveiling the unknown, distant communication networks within this system to obtain more comprehensive knowledge of how allostery impacts cardiac physiology and disease.

Pathogenesis associated with a restrictive cardiomyopathy mutant in cardiac troponin T is due to reduced protein stability and greatly increased myofilament Ca2+ sensitivity.

BACKGROUND: Dilated and hypertrophic cardiomyopathy mutations in troponin can blunt effects of protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI), decreasing myofilament Ca2+-sensitivity; however this effect has never been tested for restrictive cardiomyopathy (RCM) mutants. This study explores whether an RCM cardiac troponin T mutant (cTnT-ΔE96) interferes with convergent PKA regulation and if TnT instability contributes to greatly enhanced Ca2+-sensitivity in skinned fibers. METHODS: Force of contraction in skinned cardiac porcine fiber and spectroscopic studies were performed. RESULTS: A decrease of -0.26 and -0.25 pCa units in Ca2+-sensitivity of contraction after PKA incubation was observed for skinned fibers incorporated with WT or cTnT-ΔE96, respectively. To further assess whether cTnT-ΔE96 interferes solely with transmission of cTnI phosphorylation effects, skinned fibers were reconstituted with PKA pseudo-phosphorylated cTnI (cTnI-SS/DD.cTnC). Fibers displaced with cTnT-WT, reconstituted with cTnI-SS/DD.cTnC decreased Ca2+-sensitivity of force (pCa50=5.61) compared to control cTnI-WT.cTnC (pCa50=5.75), similarly affecting cTnT-ΔE96 (pCa50=6.03) compared to control \cTnI-WT.cTnC (pCa50=6.14). Fluorescence studies measuring cTnC(IAANS) Ca2+-affinity changes due to cTnT-ΔE96 indicated that higher complexity (thin filament) better recapitulates skinned fiber Ca2+ sensitive changes. Circular dichroism revealed reduced α-helicity and earlier thermal unfolding for cTnT-ΔE96 compared to WT. CONCLUSIONS: Although ineffective in decreasing myofilament Ca2+-sensitivity to normal levels, cTnT-ΔE96 does not interfere with PKA cTnI phosphorylation mediated effects; 2) cTnT-ΔE96 requires actin to increase cTnC Ca2+-affinity; and 3) deletion of E96 reduces cTnT stability, likely disrupting crucial thin filament interactions. GENERAL SIGNIFICANCE: The pathological effect of cTnT-ΔE96 is largely manifested by dramatic myofilament Ca2+-sensitization which still persists even after PKA phosphorylation mediated Ca2+-desensitization.

Post-translational modifications of vertebrate striated muscle myosin heavy chains.

Post-translational modifications (PTMs) play a crucial role in regulating the function of many sarcomeric proteins, including myosin. Myosins comprise a family of motor proteins that play fundamental roles in cell motility in general and muscle contraction in particular. A myosin molecule consists of two myosin heavy chains (MyHCs) and two pairs of myosin light chains (MLCs); two MLCs are associated with the neck region of each MyHC's N-terminal head domain, while the two MyHC C-terminal tails form a coiled-coil that polymerizes with other MyHCs to form the thick filament backbone. Myosin undergoes extensive PTMs, and dysregulation of these PTMs may lead to abnormal muscle function and contribute to the development of myopathies and cardiovascular disorders. Recent studies have uncovered the significance of PTMs in regulating MyHC function and showed how these PTMs may provide additional modulation of contractile processes. Here, we discuss MyHC PTMs that have been biochemically and/or functionally studied in mammals' and rodents' striated muscle. We have identified hotspots or specific regions in three isoforms of myosin (MYH2, MYH6, and MYH7) where the prevalence of PTMs is more frequent and could potentially play a significant role in fine-tuning the activity of these proteins.

A mutation in TNNC1-encoded cardiac troponin C, TNNC1-A31S, predisposes to hypertrophic cardiomyopathy and ventricular fibrillation.

Defined as clinically unexplained hypertrophy of the left ventricle, hypertrophic cardiomyopathy (HCM) is traditionally understood as a disease of the cardiac sarcomere. Mutations in TNNC1-encoded cardiac troponin C (cTnC) are a relatively rare cause of HCM. Here, we report clinical and functional characterization of a novel TNNC1 mutation, A31S, identified in a pediatric HCM proband with multiple episodes of ventricular fibrillation and aborted sudden cardiac death. Diagnosed at age 5, the proband is family history-negative for HCM or sudden cardiac death, suggesting a de novo mutation. TnC-extracted cardiac skinned fibers were reconstituted with the cTnC-A31S mutant, which increased Ca(2+) sensitivity with no effect on the maximal contractile force generation. Reconstituted actomyosin ATPase assays with 50% cTnC-A31S:50% cTnC-WT demonstrated Ca(2+) sensitivity that was intermediate between 100% cTnC-A31S and 100% cTnC-WT, whereas the mutant increased the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca(2+) affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca(2+) sensitivity of the myofilament, which may alter Ca(2+) handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.

The functional properties of human slow skeletal troponin T isoforms in cardiac muscle regulation.

Human slow skeletal troponin T (HSSTnT) shares a high degree of homology with cardiac TnT (CTnT). Although the presence of HSSTnT has not been confirmed in the heart at the protein level, detectable levels of HSSTnT mRNA have been found. Whether HSSTnT isoforms are expressed transiently remains unknown. Because transient re-expression of HSSTnT may be a potential mechanism of regulating function, we explored the effect of HSSTnT on the regulation of cardiac muscle. At least three HSSTnT isoforms have been found to exist in slow skeletal muscle: HSSTnT1 (+exons 5 and 12), HSSTnT2 (+exon 5, -exon 12), and HSSTnT3 (-exons 5 and 12). Another isoform, HSSTnT hypothetical (Hyp) (-exon 5, +exon 12), has only been found at the mRNA level. Compared with HCTnT3 (adult isoform), Tn complexes containing HSSTnT1, -2, and -3 did not alter the actomyosin ATPase activation and inhibition in the presence and absence of Ca(2+), respectively. HSSTnTHyp was not evaluated as it did not form a Tn complex under a variety of conditions. Porcine papillary skinned fibers displaced with HSSTnT1, -2, or -3 and reconstituted with human cardiac troponin I and troponin C (HCTnI·TnC) complex showed a decrease in the Ca(2+) sensitivity of force development and an increase in maximal recovered force (HSSTnT1 and -3) compared with HCTnT3. In contrast, HSSTnTHyp showed an increase in the Ca(2+) sensitivity of force development. This suggests that re- or overexpression of specific SSTnT isoforms might have therapeutic potential in the failing heart because they increase the maximal force of contraction. In addition, circular dichroism and proteolytic digestion experiments revealed structural differences between HSSTnT isoforms and HCTnT3 and that HSSTnT1 is more susceptible to calpain and trypsin proteolysis than the other HSSTnTs. Overall, HSSTnT isoforms despite being homologues of CTnT may display distinct functional properties in muscle regulation.

Strong cross-bridges potentiate the Ca(2+) affinity changes produced by hypertrophic cardiomyopathy cardiac troponin C mutants in myofilaments: a fast kinetic approach.

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca(2+) affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants. Previously, we found that incorporation of the A8V and D145E HCM-cTnC mutants, but not E134D into thin filaments (TFs), increased the apparent Ca(2+) affinity relative to TFs containing the WT protein. Here, we show that the addition of myosin subfragment 1 (S1) to TFs reconstituted with these mutants in the absence of MgATP(2-), the condition conducive to rigor cross-bridge formation, further increased the apparent Ca(2+) affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca(2+) dissociation (k(off)) from the cTnC mutants in the presence of TFs and S1. At a high level of complexity (i.e. TF + S1), an increase in the Ca(2+) affinity and decrease in k(off) was achieved for the A8V and D145E mutants when compared with WT. Therefore, it appears that the cTnC Ca(2+) off-rate is most likely to be affected rather than the Ca(2+) on rate. At all levels of TF complexity, the results obtained with the E134D mutant reproduced those seen with the WT protein. We conclude that strong cross-bridges potentiate the Ca(2+)-sensitizing effect of HCM-cTnC mutants on the myofilament. Finally, the slower k(off) from the A8V and D145E mutants can be directly correlated with the diastolic dysfunction seen in these patients.

Functional characterization of TNNC1 rare variants identified in dilated cardiomyopathy.

TNNC1, which encodes cardiac troponin C (cTnC), remains elusive as a dilated cardiomyopathy (DCM) gene. Here, we report the clinical, genetic, and functional characterization of four TNNC1 rare variants (Y5H, M103I, D145E, and I148V), all previously reported by us in association with DCM (Hershberger, R. E., Norton, N., Morales, A., Li, D., Siegfried, J. D., and Gonzalez-Quintana, J. (2010) Circ. Cardiovasc. Genet. 3, 155-161); in the previous study, two variants (Y5H and D145E) were identified in subjects who also carried MYH7 and MYBPC3 rare variants, respectively. Functional studies using the recombinant human mutant cTnC proteins reconstituted into porcine papillary skinned fibers showed decreased Ca(2+) sensitivity of force development (Y5H and M103I). Furthermore, the cTnC mutants diminished (Y5H and I148V) or abolished (M103I) the effects of PKA phosphorylation on Ca(2+) sensitivity. Only M103I decreased the troponin activation properties of the actomyosin ATPase when Ca(2+) was present. CD spectroscopic studies of apo (absence of divalent cations)-, Mg(2+)-, and Ca(2+)/Mg(2+)-bound states indicated that all of the cTnC mutants (except I148V in the Ca(2+)/Mg(2+) condition) decreased the α-helical content. These results suggest that each mutation alters the function/ability of the myofilament to bind Ca(2+) as a result of modifications in cTnC structure. One variant (D145E) that was previously reported in association with hypertrophic cardiomyopathy and that produced results in vivo in this study consistent with prior hypertrophic cardiomyopathy functional studies was found associated with the MYBPC3 P910T rare variant, likely contributing to the observed DCM phenotype. We conclude that these rare variants alter the regulation of contraction in some way, and the combined clinical, molecular, genetic, and functional data reinforce the importance of TNNC1 rare variants in the pathogenesis of DCM.

Fetal cardiac troponin isoforms rescue the increased Ca2+ sensitivity produced by a novel double deletion in cardiac troponin T linked to restrictive cardiomyopathy: a clinical, genetic, and functional approach.

A novel double deletion in cardiac troponin T (cTnT) of two highly conserved amino acids (Asn-100 and Glu-101) was found in a restrictive cardiomyopathic (RCM) pediatric patient. Clinical evaluation revealed the presence of left atrial enlargement and marked left ventricle diastolic dysfunction. The explanted heart examined by electron microscopy revealed myofibrillar disarray and mild fibrosis. Pedigree analysis established that this mutation arose de novo. The patient tested negative for six other sarcomeric genes. The single and double recombinant cTnT mutants were generated, and their functional consequences were analyzed in porcine skinned cardiac muscle. In the adult Tn environment (cTnT3 + cardiac troponin I), the single cTnT3-ΔN100 and cTnT3-ΔE101 mutations had opposing effects on the Ca(2+) sensitivity of force development compared with WT, whereas the double deletion cTnT3-ΔN100/ΔE101 increased the Ca(2+) sensitivity + 0.19 pCa units. In addition, cTnT3-ΔN100/ΔE101 decreased the cooperativity of force development, suggesting alterations in intrafilament protein-protein interactions. In the fetal Tn environment, (cTnT1 + slow skeletal troponin I), the single (cTnT1-ΔN110) and double (cTnT1-ΔN110/ΔE111) deletions did not change the Ca(2+) sensitivity compared with control. To recreate the patient's heterozygous genotype, we performed a reconstituted ATPase activity assay. Thin filaments containing 50:50 cTnT3-ΔN100/ΔE101:cTnT3-WT also increased the myofilament Ca(2+) sensitivity compared with WT. Co-sedimentation of thin filament proteins indicated that no significant changes occurred in the binding of Tn containing the RCM cTnT mutation to actin-Tm. This report reveals the protective role of Tn fetal isoforms as they rescue the increased Ca(2+) sensitivity produced by a cTnT-RCM mutation and may account for the lack of lethality during gestation.

Autoimmunity as a sequela to obesity and systemic inflammation.

The rising prevalence of obesity presents a world-wide challenge as it is associated with numerous comorbidities including cardiovascular disease, insulin resistance and hypertension. Obesity-associated illnesses are estimated to cause nearly 4 million deaths globally per year, therefore there is a critical need to better understand associated pathogenesis, identify new therapeutic targets, and develop new interventions. Emerging data identify a key role for chronic inflammation in mediating obesity related disease states and reveal higher incidence of autoimmune disease development. Of the multiple potential mechanisms linking obesity and autoimmunity, the strongest link has been shown for leptin, a hormone secreted at high levels from obese white adipose tissue. Numerous studies have demonstrated that leptin enhances activation of both arms of the immune system, while its absence protects against development of autoimmunity. Other potential newly discovered mechanisms that contribute to autoimmune pathogenesis are not directly connected but also associated with obesity including sustained platelet activation, gut dysbiosis, and aging. Here we review how obesity instigates autoimmunity, particularly in the context of immune cell activations and adipokine secretion.

Post-translational modification patterns on ß-myosin heavy chain are altered in ischemic and nonischemic human hearts.

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 ß-myosin heavy chain (ß-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 ß-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 ß-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.

Predicting cardiomyopathic phenotypes by altering Ca2+ affinity of cardiac troponin C.

Cardiac diseases associated with mutations in troponin subunits include hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM). Altered calcium handling in these diseases is evidenced by changes in the Ca(2+) sensitivity of contraction. Mutations in the Ca(2+) sensor, troponin C (TnC), were generated to increase/decrease the Ca(2+) sensitivity of cardiac skinned fibers to create the characteristic effects of DCM, HCM, and RCM. We also used a reconstituted assay to determine the mutation effects on ATPase activation and inhibition. One mutant (A23Q) was found with HCM-like properties (increased Ca(2+) sensitivity of force and normal levels of ATPase inhibition). Three mutants (S37G, V44Q, and L48Q) were identified with RCM-like properties (a large increase in Ca(2+) sensitivity, partial loss of ATPase inhibition, and increased basal force). Two mutations were identified (E40A and I61Q) with DCM properties (decreased Ca(2+) sensitivity, maximal force recovery, and activation of the ATPase at high [Ca(2+)]). Steady-state fluorescence was utilized to assess Ca(2+) affinity in isolated cardiac (c)TnCs containing F27W and did not necessarily mirror the fiber Ca(2+) sensitivity. Circular dichroism of mutant cTnCs revealed a trend where increased alpha-helical content correlated with increased Ca(2+) sensitivity in skinned fibers and vice versa. The main findings from this study were as follows: 1) cTnC mutants demonstrated distinct functional phenotypes reminiscent of bona fide HCM, RCM, and DCM mutations; 2) a region in cTnC associated with increased Ca(2+) sensitivity in skinned fibers was identified; and 3) the F27W reporter mutation affected Ca(2+) sensitivity, maximal force, and ATPase activation of some mutants.

Cardiovascular Injury Due to SARS-CoV-2.

Purpose of Review: The world is currently facing the largest global health crisis since the early 1900s due to a novel coronavirus. While SARS-CoV-2 infection causes predictable symptoms in COVID-19 patients, including upper respiratory distress and fever, the heterogeneity of manifestations is surprising. This review focuses on direct and indirect causes of myocardial injury in COVID-19 patients and highlights current knowledge, treatment strategies, and outstanding questions in the field. Recent Findings: Data are emerging that highlight the extent of cardiovascular involvement in COVID-19 patients, including evidence that SARS-CoV-2 causes myocarditis and increases cardiac risk. The incidence of cardiac injury is much greater in patients with severe disease presentation and those in intensive care. Summary: During the past year, COVID-19 patient mortality rates have improved due to tailored pharmacological treatments and patient management strategies that address the unique presentation of symptoms, which will hopefully also reduce the incidence of cardiac injury.

Essential roles of the dystrophin-glycoprotein complex in different cardiac pathologies.

The dystrophin-glycoprotein complex (DGC), situated at the sarcolemma dynamically remodels during cardiac disease. This review examines DGC remodeling as a common denominator in diseases affecting heart function and health. Dystrophin and the DGC serve as broad cytoskeletal integrators that are critical for maintaining stability of muscle membranes. The presence of pathogenic variants in genes encoding proteins of the DGC can cause absence of the protein and/or alterations in other complex members leading to muscular dystrophies. Targeted studies have allowed the individual functions of affected proteins to be defined. The DGC has demonstrated its dynamic function, remodeling under a number of conditions that stress the heart. Beyond genetic causes, pathogenic processes also impinge on the DGC, causing alterations in the abundance of dystrophin and associated proteins during cardiac insult such as ischemia-reperfusion injury, mechanical unloading, and myocarditis. When considering new therapeutic strategies, it is important to assess DGC remodeling as a common factor in various heart diseases. The DGC connects the internal F-actin-based cytoskeleton to laminin-211 of the extracellular space, playing an important role in the transmission of mechanical force to the extracellular matrix. The essential functions of dystrophin and the DGC have been long recognized. DGC based therapeutic approaches have been primarily focused on muscular dystrophies, however it may be a beneficial target in a number of disorders that affect the heart. This review provides an account of what we now know, and discusses how this knowledge can benefit persistent health conditions in the clinic.

Anomalous structural dynamics of minimally frustrated residues in cardiac troponin C triggers hypertrophic cardiomyopathy.

Cardiac TnC (cTnC) is highly conserved among mammals, and genetic variants can result in disease by perturbing Ca2+-regulation of myocardial contraction. Here, we report the molecular basis of a human mutation in cTnC's αD-helix (TNNC1-p.C84Y) that impacts conformational dynamics of the D/E central-linker and sampling of discrete states in the N-domain, favoring the "primed" state associated with Ca2+ binding. We demonstrate cTnC's αD-helix normally functions as a central hub that controls minimally frustrated interactions, maintaining evolutionarily conserved rigidity of the N-domain. αD-helix perturbation remotely alters conformational dynamics of the N-domain, compromising its structural rigidity. Transgenic mice carrying this cTnC mutation exhibit altered dynamics of sarcomere function and hypertrophic cardiomyopathy. Together, our data suggest that disruption of evolutionary conserved molecular frustration networks by a myofilament protein mutation may ultimately compromise contractile performance and trigger hypertrophic cardiomyopathy.