Vikram Shettigar

Rationally engineered Troponin C modulates in vivo cardiac function and performance in health and disease

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 Ca2+ signal. Promisingly, our smartly formulated Ca2+-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.

Engineering Parvalbumin for the Heart: Optimizing the Mg2+ Binding Properties of Rat β-Parvalbumin

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 Ca2+ dissociation and are prone to becoming saturated with Ca2+, 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 Mg2+ affinity is needed. We demonstrate that at 35°C, rat β-PV has an ~30-fold faster rate of Ca2+ dissociation compared to rat skeletal muscle α-PV, and still possesses a physiologically relevant Ca2+ affinity (~100 nM). However, rat β-PV will not be a delayed Ca2+ buffer since its Mg2+ affinity is too low (~1 mM). We have engineered two mutations into rat β-PV, S55D and E62D, when observed alone increase Mg2+ affinity up to fivefold, but when combined increase Mg2+ affinity ~13-fold, well within a physiologically relevant affinity. Furthermore, the Mg2+ dissociation rate (172/s) from the engineered S55D, E62D PV is slow enough for delayed Ca2+ buffering. Additionally, the engineered PV retains a high Ca2+ affinity (132 nM) and fast rate of Ca2+ 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.

Designing proteins to combat disease: Cardiac troponin C as an example.

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.

In Utero Particulate Matter Exposure Produces Heart Failure, Electrical Remodeling, and Epigenetic Changes at Adulthood

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/m3 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/dtmax/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 Ca2+‐ATPase, phosphorylated phospholamban, and Na+/Ca2+ exchanger were decreased. Conclusions In utero PM 2.5 exposure triggers an acute inflammatory response, chronic matrix remodeling, and alterations in Ca2+ handling proteins, resulting in global adult cardiac dysfunction. These results also highlight the potential involvement of epigenetics in priming of adult cardiac disease.

TGF-β1 affects cell-cell adhesion in the heart in an NCAM1-dependent mechanism

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.

The RhoGAP Myo9b Promotes Bone Growth by Mediating Osteoblastic Responsiveness to IGF-1†

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.

Divergent Soybean Calmodulins Respond Similarly to Calcium Transients: Insight into Differential Target Regulation

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 Mg2+ and the CaM binding domains from proteins that are differentially regulated, the two plant CaMs respond nearly identically to rapid and slow Ca2+ transients. Our data suggest that the plant CaMs have evolved to bind certain targets with comparable affinities, respond similarly to a particular Ca2+ 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.

NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model

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.The molecular mechanisms leading to heart failure in patients with Duchenne muscular dystrophy are unclear. Here the authors show that NF-κB is activated in the heart of dystrophin-deficient mice and that its ablation rescues cardiac function through chromatin remodeling and activation of gene expression.

Gene Transfer of Engineered Calmodulin Alleviates Ventricular Arrhythmias in a Calsequestrin‐Associated Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia

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.