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Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology. Keywords: antisense oligonucleotides, myosin, cardiac hypertrophy, cardiomyopathy, nucleic acid, RNA degradation

The identity of the specific nitric oxide dioxygenase (NOD) that serves as the main in vivo regulator of O2-dependent NO degradation in smooth muscle remains elusive. Cytoglobin (Cygb) is a recently discovered globin expressed in fibroblasts and smooth muscle cells with unknown function. Cygb, coupled with a cellular reducing system, efficiently regulates the rate of NO consumption by metabolizing NO in an O2-dependent manner with decreased NO consumption in physiological hypoxia. Here we show that Cygb is a major regulator of NO degradation and cardiovascular tone. Knockout of Cygb greatly prolongs NO decay, increases vascular relaxation, and lowers blood pressure and systemic vascular resistance. We further demonstrate that downregulation of Cygb prevents angiotensin-mediated hypertension. Thus, Cygb has a critical role in the regulation of vascular tone and disease. We suggest that modulation of the expression and NOD activity of Cygb represents a strategy for the treatment of cardiovascular disease., The gaseous signalling molecule nitric oxide regulates vascular tone. Here, the authors show that nitric oxide is degraded by the enzyme cytoglobin in the vascular wall, and that mice lacking cytoglobin have reduced blood pressure and are less sensitive to angiotensin-mediated hypertension.

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.

Visual Abstract, Highlights • The pathogenesis of human heart failure is complex, and the creation of new therapeutic strategies for human heart failure is critical. • Identifying the molecular pathways underlying heart failure is important to define potential new therapeutic targets. • Ankyrin polypeptides serve to target and stabilize membrane proteins in cardiomyocytes. • Ankyrin-G levels are altered in humans and mice with heart failure, and mice lacking ankyrin-G in cardiomyocytes develop cardiomyopathy and systolic dysfunction. • Mechanistically, ankyrin-G is necessary for the expression and localization of critical myocyte proteins essential for regulating cardiac structural and electrical activity., Summary Ankyrin polypeptides are intracellular proteins responsible for targeting cardiac membrane proteins. Here, the authors demonstrate that ankyrin-G plays an unexpected role in normal compensatory physiological remodeling in response to myocardial stress and aging; the authors implicate disruption of ankyrin-G in human heart failure. Mechanistically, the authors illustrate that ankyrin-G serves as a key nodal protein required for cardiac myofilament integration with the intercalated disc. Their data define novel in vivo mechanistic roles for ankyrin-G, implicate ankyrin-G as necessary for compensatory cardiac physiological remodeling under stress, and implicate disruption of ankyrin-G in the development and progression of human heart failure.

Proper trafficking of membrane-bound ion channels and transporters is requisite for normal cardiac function. Endosome-based protein trafficking of membrane-bound ion channels and transporters in the heart is poorly understood, particularly in vivo. In fact, for select cardiac cell types such as atrial myocytes, virtually nothing is known regarding endosomal transport. We previously linked the C-terminal Eps15 homology domain-containing protein 3 (EHD3) with endosome-based protein trafficking in ventricular cardiomyocytes. Here we sought to define the roles and membrane protein targets for EHD3 in atria. We identify the voltage-gated T-type Ca2+ channels (CaV3.1, CaV3.2) as substrates for EHD3-dependent trafficking in atria. Mice selectively lacking EHD3 in heart display reduced expression and targeting of both Cav3.1 and CaV3.2 in the atria. Furthermore, functional experiments identify a significant loss of T-type-mediated Ca2+ current in EHD3-deficient atrial myocytes. Moreover, EHD3 associates with both CaV3.1 and CaV3.2 in co-immunoprecipitation experiments. T-type Ca2+ channel function is critical for proper electrical conduction through the atria. Consistent with these roles, EHD3-deficient mice demonstrate heart rate variability, sinus pause, and atrioventricular conduction block. In summary, our findings identify CaV3.1 and CaV3.2 as substrates for EHD3-dependent protein trafficking in heart, provide in vivo data on endosome-based trafficking pathways in atria, and implicate EHD3 as a key player in the regulation of atrial myocyte excitability and cardiac conduction. Background: Endosome-based protein trafficking in the heart is poorly understood. Results: Functional targeting and expression of the T-type Ca2+ channel (TTCC) in the atria requires the endosomal protein, EHD3. Conclusion: Impaired endosomal transport leads to cardiac rhythm and conduction disorders. Significance: Understanding endosome-based protein trafficking in the heart may provide new therapeutic targets for cardiovascular disease.

Rationale: Na v 1.5 ( SCN5A ) is the primary cardiac voltage-gated Na v channel. Na v 1.5 is critical for cardiac excitability and conduction, and human SCN5A mutations cause sinus node dysfunction, atrial fibrillation, conductional abnormalities, and ventricular arrhythmias. Further, defects in Na v 1.5 regulation are linked with malignant arrhythmias associated with human heart failure. Consequently, therapies to target select Na v 1.5 properties have remained at the forefront of cardiovascular medicine. However, despite years of investigation, the fundamental pathways governing Na v 1.5 membrane targeting, assembly, and regulation are still largely undefined. Objective: Define the in vivo mechanisms underlying Na v 1.5 membrane regulation. Methods and Results: Here, we define the molecular basis of an Na v channel regulatory platform in heart. Using new cardiac-selective ankyrin-G -/- mice (conditional knock-out mouse), we report that ankyrin-G targets Na v 1.5 and its regulatory protein calcium/calmodulin–dependent kinase II to the intercalated disc. Mechanistically, β IV -spectrin is requisite for ankyrin-dependent targeting of calcium/calmodulin–dependent kinase II-δ; however, β IV -spectrin is not essential for ankyrin-G expression. Ankyrin-G conditional knock-out mouse myocytes display decreased Na v 1.5 expression/membrane localization and reduced I Na associated with pronounced bradycardia, conduction abnormalities, and ventricular arrhythmia in response to Na v channel antagonists. Moreover, we report that ankyrin-G links Na v channels with broader intercalated disc signaling/structural nodes, as ankyrin-G loss results in reorganization of plakophilin-2 and lethal arrhythmias in response to β-adrenergic stimulation. Conclusions: Our findings provide the first in vivo data for the molecular pathway required for intercalated disc Na v 1.5 targeting/regulation in heart. Further, these new data identify the basis of an in vivo cellular platform critical for membrane recruitment and regulation of Na v 1.5.

The binding of Ca 2 + to troponin C (TnC) in the troponin complex is a critical step regulating the thin filament, the actin–myosin interaction and cardiac contraction. Phosphorylation of the troponin complex is a key regulatory mechanism to match cardiac contraction to demand. Here we demonstrate that phosphorylation of the troponin I (TnI) subunit is simultaneously increased at Ser-150 and Ser-23/24 during in vivo myocardial ischemia. Myocardial ischemia decreases intracellular pH resulting in depressed binding of Ca 2 + to TnC and impaired contraction. To determine the pathological relevance of these simultaneous TnI phosphorylations we measured individual TnI Ser-150 (S150D), Ser-23/24 (S23/24D) and combined (S23/24/150D) pseudo-phosphorylation effects on thin filament regulation at acidic pH similar to that in myocardial ischemia. Results demonstrate that while acidic pH decreased thin filament Ca 2 + binding to TnC regardless of TnI composition, TnI S150D attenuated this decrease rendering it similar to non-phosphorylated TnI at normal pH. The dissociation of Ca 2 + from TnC was unaltered by pH such that TnI S150D remained slow, S23/24D remained accelerated and the combined S23/24/150D remained accelerated. This effect of the combined TnI Ser-150 and Ser-23/24 pseudo-phosphorylations to maintain Ca 2 + binding while accelerating Ca 2 + dissociation represents the first post-translational modification of troponin by phosphorylation to both accelerate thin filament deactivation and maintain Ca 2 + sensitive activation. These data suggest that TnI Ser-150 phosphorylation induced attenuation of the pH-dependent decrease in Ca 2 + sensitivity and its combination with Ser-23/24 phosphorylation to maintain accelerated thin filament deactivation may impart an adaptive role to preserve contraction during acidic ischemia pH without slowing relaxation.

Rationale : Cardiac function is dependent on the coordinate activities of membrane ion channels, transporters, pumps, and hormone receptors to tune the membrane electrochemical gradient dynamically in response to acute and chronic stress. Although our knowledge of membrane proteins has rapidly advanced during the past decade, our understanding of the subcellular pathways governing the trafficking and localization of integral membrane proteins is limited and essentially unstudied in vivo. In the heart, to our knowledge, there are no in vivo mechanistic studies that directly link endosome-based machinery with cardiac physiology. Objective : To define the in vivo roles of endosome-based cellular machinery for cardiac membrane protein trafficking, myocyte excitability, and cardiac physiology. Methods and Results : We identify the endosome-based Eps15 homology domain 3 (EHD3) pathway as essential for cardiac physiology. EHD3-deficient hearts display structural and functional defects including bradycardia and rate variability, conduction block, and blunted response to adrenergic stimulation. Mechanistically, EHD3 is critical for membrane protein trafficking, because EHD3-deficient myocytes display reduced expression/localization of Na/Ca exchanger and L-type Ca channel type 1.2 with a parallel reduction in Na/Ca exchanger–mediated membrane current and Ca v 1.2-mediated membrane current. Functionally, EHD3-deficient myocytes show increased sarcoplasmic reticulum [Ca], increased spark frequency, and reduced expression/localization of ankyrin-B, a binding partner for EHD3 and Na/Ca exchanger. Finally, we show that in vivo EHD3-deficient defects are attributable to cardiac-specific roles of EHD3 because mice with cardiac-selective EHD3 deficiency demonstrate both structural and electric phenotypes. Conclusions : These data provide new insight into the critical role of endosome-based pathways in membrane protein targeting and cardiac physiology. EHD3 is a critical component of protein trafficking in heart and is essential for the proper membrane targeting of select cellular proteins that maintain excitability.

Enzyme-linked and electrochemiluminescence immunoassays were developed for quantification of amino (N-) terminal fragments of the skeletal muscle protein titin (N-ter titin) and qualified for use in detection of urinary N-ter titin excretion. Urine from normal subjects contained a small but measurable level of N-ter titin (1.0 ± 0.4 ng/ml). A 365-fold increase (365.4 ± 65.0, P = 0.0001) in urinary N-ter titin excretion was seen in Duchene muscular dystrophy (DMD) patients. Urinary N-ter titin was also evaluated in dystrophin deficient rodent models. Mdx mice exhibited low urinary N-ter titin levels at 2 weeks of age followed by a robust and sustained elevation starting at 3 weeks of age, coincident with the development of systemic skeletal muscle damage in this model; fold elevation could not be determined because urinary N-ter titin was not detected in age-matched wild type mice. Levels of serum creatine kinase and serum skeletal muscle troponin I (TnI) were also low at 2 weeks, elevated at later time points and were significantly correlated with urinary N-ter titin excretion in mdx mice. Corticosteroid treatment of mdx mice resulted in improved exercise performance and lowering of both urinary N-ter titin and serum skeletal muscle TnI concentrations. Low urinary N-ter titin levels were detected in wild type rats (3.0 ± 0.6 ng/ml), while Dmdmdx rats exhibited a 556-fold increase (1652.5 ± 405.7 ng/ml, P = 0.002) (both at 5 months of age). These results suggest that urinary N-ter titin is present at low basal concentrations in normal urine and increases dramatically coincident with muscle damage produced by dystrophin deficiency. Urinary N-ter titin has potential as a facile, non-invasive and translational biomarker for DMD.

Kinase/phosphatase balance governs cardiac excitability in health and disease. Although detailed mechanisms for cardiac kinase regulation are established, far less is known regarding cardiac protein phosphatase 2A (PP2A) regulation. This is largely due to the complexity of the PP2A holoenzyme structure (combinatorial assembly of three subunit enzyme from >17 subunit genes) and the inability to segregate "global" PP2A function from the activities of multiple "local" holoenzyme populations. Here we report that PP2A catalytic, regulatory, and scaffolding subunits are tightly regulated at transcriptional, translational, and post-translational levels to tune myocyte function at base line and in disease. We show that past global read-outs of cellular PP2A activity more appropriately represent the collective activity of numerous individual PP2A holoenzymes, each displaying a specific subcellular localization (dictated by select PP2A regulatory subunits) as well as local specific post-translational catalytic subunit methylation and phosphorylation events that regulate local and rapid holoenzyme assembly/disassembly (via leucine carboxymethyltransferase 1/phosphatase methylesterase 1 (LCMT-1/PME-1). We report that PP2A subunits are selectively regulated between human and animal models, across cardiac chambers, and even within specific cardiac cell types. Moreover, this regulation can be rapidly tuned in response to cellular activation. Finally, we report that global PP2A is altered in human and experimental models of heart disease, yet each pathology displays its own distinct molecular signature though specific PP2A subunit modulatory events. These new data provide an initial view into the signaling pathways that govern PP2A function in heart but also establish the first step in defining specific PP2A regulatory targets in health and disease.

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme central to the regulation of metabolic homeostasis. In the heart AMPK is activated during cardiac stress-induced ATP depletion and functions to stimulate metabolic pathways that restore the AMP/ATP balance. Recently it was demonstrated that AMPK phosphorylates cardiac troponin I (cTnI) at Ser-150 in vitro. We sought to determine if the metabolic regulatory kinase AMPK phosphorylates cTnI at Ser-150 in vivo to alter cardiac contractile function directly at the level of the myofilament. Rabbit cardiac myofibrils separated by two-dimensional isoelectric focusing subjected to a Western blot with a cTnI phosphorylation-specific antibody demonstrates that cTnI is endogenously phosphorylated at Ser-150 in the heart. Treatment of myofibrils with the AMPK holoenzyme increased cTnI Ser-150 phosphorylation within the constraints of the muscle lattice. Compared with controls, cardiac fiber bundles exchanged with troponin containing cTnI pseudo-phosphorylated at Ser-150 demonstrate increased sensitivity of calcium-dependent force development, blunting of both PKA-dependent calcium desensitization, and PKA-dependent increases in length dependent activation. Thus, in addition to the defined role of AMPK as a cardiac metabolic energy gauge, these data demonstrate AMPK Ser-150 phosphorylation of cTnI directly links the regulation of cardiac metabolic demand to myofilament contractile energetics. Furthermore, the blunting effect of cTnI Ser-150 phosphorylation cross-talk can uncouple the effects of myofilament PKA-dependent phosphorylation from β-adrenergic signaling as a novel thin filament contractile regulatory signaling mechanism.

The calcium-dependent interactions between troponin C (TnC) and other thin and thick filament proteins play a key role in the regulation of cardiac muscle contraction. Five hydrophobic residues (Phe(20), Val(44), Met(45), Leu(48), and Met(81)) in the regulatory domain of TnC were individually substituted with polar Gln, to examine the effect of these mutations that sensitized isolated TnC to calcium on (1) the calcium binding and exchange with TnC in increasingly complex biochemical systems and (2) the calcium sensitivity of actomyosin ATPase. The hydrophobic residue mutations drastically affected calcium binding and exchange with TnC in increasingly complex biochemical systems, indicating that side chain intra- and intermolecular interactions of these residues play a crucial role in determining how TnC responds to calcium. However, the mutations that sensitized isolated TnC to calcium did not necessarily increase the calcium sensitivity of the troponin (Tn) complex or reconstituted thin filaments with or without myosin S1. Furthermore, the calcium sensitivity of reconstituted thin filaments (in the absence of myosin S1) was a better predictor of the calcium dependence of actomyosin ATPase activity than that of TnC or the Tn complex. Thus, both the intrinsic properties of TnC and its interactions with the other contractile proteins play a crucial role in modulating the binding of calcium to TnC in increasingly complex biochemical systems.

Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme composed of a catalytic, scaffolding, and regulatory subunit. In the heart, PP2A activity is requisite for cardiac excitation-contraction coupling and central in adrenergic signaling. We found that mice deficient in the PP2A regulatory subunit B56α (1 of 13 regulatory subunits) had altered PP2A signaling in the heart that was associated with changes in cardiac physiology, suggesting that the B56α regulatory subunit had an autoinhibitory role that suppressed excess PP2A activity. The increase in PP2A activity in the mice with reduced B56α expression resulted in slower heart rates and increased heart rate variability, conduction defects, and increased sensitivity of heart rate to parasympathetic agonists. Increased PP2A activity in B56α(+/-) myocytes resulted in reduced Ca(2+) waves and sparks, which was associated with decreased phosphorylation (and thus decreased activation) of the ryanodine receptor RyR2, an ion channel on intracellular membranes that is involved in Ca(2+) regulation in cardiomyocytes. In line with an autoinhibitory role for B56α, in vivo expression of B56α in the absence of altered abundance of other PP2A subunits decreased basal phosphatase activity. Consequently, in vivo expression of B56α suppressed parasympathetic regulation of heart rate and increased RyR2 phosphorylation in cardiomyocytes. These data show that an integral component of the PP2A holoenzyme has an important inhibitory role in controlling PP2A enzyme activity in the heart.

Background— Voltage-gated Na + channels (Na v ) are essential for myocyte membrane excitability and cardiac function. Na v current ( I Na ) is a large-amplitude, short-duration spike generated by rapid channel activation followed immediately by inactivation. However, even under normal conditions, a small late component of I Na ( I Na,L ) persists because of incomplete/failed inactivation of a subpopulation of channels. Notably, I Na,L is directly linked with both congenital and acquired disease states. The multifunctional Ca 2+ /calmodulin-dependent kinase II (CaMKII) has been identified as an important activator of I Na,L in disease. Several potential CaMKII phosphorylation sites have been discovered, including Ser571 in the Na v 1.5 DI-DII linker, but the molecular mechanism underlying CaMKII-dependent regulation of I Na,L in vivo remains unknown. Methods and Results— To determine the in vivo role of Ser571, 2 Scn5a knock-in mouse models were generated expressing either: (1) Na v 1.5 with a phosphomimetic mutation at Ser571 (S571E), or (2) Na v 1.5 with the phosphorylation site ablated (S571A). Electrophysiology studies revealed that Ser571 regulates I Na,L but not other channel properties previously linked to CaMKII. Ser571-mediated increases in I Na,L promote abnormal repolarization and intracellular Ca 2+ handling and increase susceptibility to arrhythmia at the cellular and animal level. Importantly, Ser571 is required for maladaptive remodeling and arrhythmias in response to pressure overload. Conclusions— Our data provide the first in vivo evidence for the molecular mechanism underlying CaMKII activation of the pathogenic I Na,L . Relevant for improved rational design of potential therapies, our findings demonstrate that Ser571-dependent regulation of Na v 1.5 specifically tunes I Na,L without altering critical physiological components of the current.

Background— The cardiac cytoskeleton plays key roles in maintaining myocyte structural integrity in health and disease. In fact, human mutations in cardiac cytoskeletal elements are tightly linked to cardiac pathologies, including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction and cardiac electric activity is not well understood and often overlooked in the cardiac arrhythmia field. Methods and Results— Here, we uncover a new mechanism for the regulation of cardiac membrane excitability. We report that βII spectrin, an actin-associated molecule, is essential for the posttranslational targeting and localization of critical membrane proteins in heart. βII spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/βII spectrin interaction, leading to severe human arrhythmia phenotypes. Mice lacking cardiac βII spectrin display lethal arrhythmias, aberrant electric and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, βII spectrin regulates the localization of cytoskeletal and plasma membrane/sarcoplasmic reticulum protein complexes, including the Na/Ca exchanger, ryanodine receptor 2, ankyrin-B, actin, and αII spectrin. Finally, we observe accelerated heart failure phenotypes in βII spectrin–deficient mice. Conclusions— Our findings identify βII spectrin as critical for normal myocyte electric activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology.

Excessive oxidative stress in the heart results in contractile dysfunction. While antioxidant therapies have been a disappointment clinically, exercise has shown beneficial results, in part by reducing oxidative stress. We have previously shown that neuronal nitric oxide synthase (nNOS) is essential for cardioprotective adaptations caused by exercise. We hypothesize that part of the cardioprotective role of nNOS is via the augmentation of the antioxidant defense with exercise by positively shifting the nitroso-redox balance. Our results show that nNOS is indispensable for the augmented anti-oxidant defense with exercise. Furthermore, exercise training of nNOS knockout mice resulted in a negative shift in the nitroso-redox balance resulting in contractile dysfunction. Remarkably, overexpressing nNOS (conditional cardiac-specific nNOS overexpression) was able to mimic exercise by increasing VO2max. This study demonstrates that exercise results in a positive shift in the nitroso-redox balance that is nNOS-dependent. Thus, targeting nNOS signaling may mimic the beneficial effects of exercise by combating oxidative stress and may be a viable treatment strategy for heart disease.

We have engineered cardiac TnCs with increased (L48Q) or decreased (D73N) Ca2+ sensitivity. To express these proteins in the in vivo heart we utilized an adeno-associated virus serotype 9 (AAV-9). The Ca2+ desensitized D73N TnC recapitulated a dilated cardiomyopathy phenotype and depressed function as observed by echocardiography and isolated cardiomyocytes. On the other hand, AAV-9 containing the Ca2+ sensitized L48Q TnC did not cause any disease phenotype or arrhythmias commonly associated with increased myofilament Ca2+ sensitivity. In healthy mice, L48Q TnC increased myocyte contraction and whole heart contractility with improved cardiovascular performance (increased V02max). Excitingly, L48Q TnC expressing mice were able to preserve higher contractility, ejection fraction, cardiac performance and decreased death rate even after undergoing trans-aortic constriction or myocardial infarction. Additionally, L48Q TnC was able to increase contractility, ejection fraction and cardiac performance in mice which expressed L48Q TnC after having a myocardial infarction. In summary, engineered TnCs show potential to be used as treatment strategies against different cardiomyopathies.

The sinoatrial node (SAN) is the intrinsic pacemaker of the vertebrate heart, which is responsible for the generation and modulation of cardiac automaticity. Dysfunction of pacemaking function is associated with a host of human arrhythmia phenotypes termed sinus node disease or “sick sinus syndrome.” Sinus node disease may include phenotypes of sinus bradycardia, sinus node exit block, sinus node pause/arrest, and instances of atrial brady/tachyarrhythmias. 1 Sinus node disease affects hundreds of thousands of individuals in the United States annually and results in tens of thousands of patients requiring pacemaker therapy each year. While SAN-linked arrhythmias may occur in children with congenital forms of sick sinus syndrome, phenotypes can occur at any age, although the prevalence increases with age. In fact, 1 of every 600 patients with cardiac disease who are older than 65 years requires pacemaker implantation. 2 Previous work has suggested that the age-dependent decline of SAN function, symptoms that include a decrease in intrinsic heart rate and increase in action potential duration (APD), could be attributed to fibrosis. 3,4 However, more recent evidence links dysfunction in ion channel and transporter regulation in the pathogenesis of SAN disease. Over the last 2 decades, our understanding of SAN ion channel/transporter expression and modulation has dramatically increased. The ability of the SAN to generate spontaneous electrical activity is dependent on the synchronized activation of SAN ion channels and transporters. SAN diastolic depolarization is generated by the hyperpolarization-activated cyclic nucleotide-gated (HCN4) channel and the Na/Ca exchanger (NCX). Once the SAN cell membrane potential reaches the threshold potential of both L- and T-type calcium channels, the action potential (AP) is modulated by a host of additional membrane and sarcoplasmic reticulum (SR) Ca 2þ regulatory

A hallmark of cardiac ischemia is decreased intracellular pH which can affect a number of cellular processes. Such an acidic environment alters cardiac troponin (Tn) myofilament regulation to decrease the Ca2+ sensitivity of force production. Tn also undergoes cardiac ischemic-induced AMPK troponin I (TnI) Ser-150 phosphorylation. We recently characterized the effect of TnI Ser-150 phosphorylation demonstrating it to increase myofilament Ca2+ sensitivity and blunt TnI PKA phosphorylation-induced desensitization. The effect of an acidic environment on Tn myofilament regulation in the presence of the ischemic-induced TnI Ser-150 phosphorylation is unknown. In this study we investigate the effect of pH on myofilament regulation in the presence of TnI Ser-150 phosphorylation. Myofilament regulation was determined by measuring troponin C Ca2+ binding properties at normal and acidic pH. Results demonstrate acidic pH decreases steady-state Ca2+ binding to troponin C in thin filaments reconstituted with either WT or Ser-150 pseudo-phosphorylated TnI (Tn S150D) such that Tn S150D Ca2+ sensitivity at pH 6.5 is similar to WT at pH 7. Furthermore, while Tn S150D slowed Ca2+ dissociation compared to Tn WT at pH 7.0, decreasing the pH to 6.5 did not further alter isolated Tn S150D Ca2+ dissociation indicating a lack of pH to modulate Tn Ca2+ dissociation kinetics. Thin filament dissociation kinetics are ongoing. We conclude that TnI Ser-150 phosphorylation imparts resistance to acidic pH induced myofilament Ca2+ desensitization similar to that present during ischemia without alteration of Tn Ca2+ dissociation. Future investigations are aimed at examining the effect of TnI Ser-150 phosphorylation in the presence of TnI Ser-23/24 phosphorylation at low pH.

The Ca(2+) binding properties of the FHC-associated cardiac troponin C (cTnC) mutation L29Q were examined in isolated cTnC, troponin complexes, reconstituted thin filament preparations, and skinned cardiomyocytes. While higher Ca(2+) binding affinity was apparent for the L29Q mutant in isolated cTnC, this phenomenon was not observed in the cTn complex. At the level of the thin filament in the presence of phosphomimetic TnI, L29Q cTnC further reduced the Ca(2+) affinity by 27% in the steady-state measurement and increased the Ca(2+) dissociation rate by 20% in the kinetic studies. Molecular dynamics simulations suggest that L29Q destabilizes the conformation of cNTnC in the presence of phosphomimetic cTnI and potentially modulates the Ca(2+) sensitivity due to the changes of the opening/closing equilibrium of cNTnC. In the skinned cardiomyocyte preparation, L29Q cTnC increased Ca(2+) sensitivity in a highly sarcomere length (SL)-dependent manner. The well-established reduction of Ca(2+) sensitivity by phosphomimetic cTnI was diminished by 68% in the presence of the mutation and it also depressed the SL-dependent increase in myofilament Ca(2+) sensitivity. This might result from its modified interaction with cTnI which altered the feedback effects of cross-bridges on the L29Q cTnC-cTnI-Tm complex. This study demonstrates that the L29Q mutation alters the contractility and the functional effects of the phosphomimetic cTnI in both thin filament and single skinned cardiomyocytes and importantly that this effect is highly sarcomere length dependent.

The troponin (Tn) complex is a critical regulatory and integrative hub for myofilament post-translational modifications that regulate cardiac contraction. Beta-adrenergic-induced protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI) at Ser-23/24 is a major myofilament cardiac contractile modulator. In addition to this PKA-induced phosphorylation, cTnI can be simultaneously phosphorylated at a number of other residues. To fully understand the molecular mechanisms that underlie cardiac muscle regulation at the myofilament level, cTnI phosphorylations must be studied as integrated events. As an initial step towards understanding the integration of these events, we employed a phosphomimetic approach to investigate the effect of cTnI Ser-150 pseudo-phosphorylation (TnI-S150D) on cTnI PKA pseudo-phosphorylation (TnI-S23/24D) function within the thin filament. The combinatorial effect of these phosphorylation events were determined by measuring the binding of calcium to troponin C in thin filaments reconstituted with human Tn. The steady-state calcium sensitivity of TnC was increased ∼2.3 fold in thin filaments reconstituted with TnI-S150D containing Tn, while TnI-S23/24D containing filaments exhibited an ∼3.9 fold decrease of calcium sensitivity. Importantly, in the presence of both cTnI pseudo-phosphorylations (TnI-S23/24/150D) calcium binding was not different from that of wild-type. Likewise, calcium dissociation from thin filaments reconstituted with TnI-S23/24D was increased by ∼3.5 fold, while TnI-S150D containing filaments demonstrated a decrease in calcium dissociation of ∼2.3 fold compared to wild-type filaments. Interestingly the combined pseudo-phosphorylations of TnI-S23/24/150D only blunted TnI-23/24D calcium dissociation kinetics to ∼1.2 fold of the TnI-S23/24D filaments alone. These findings demonstrate the integrated effects of cTnI site-specific phosphorylation crosstalk to affect the function of cTnI PKA-induced phosphorylation. These data further highlight the importance of understanding the integrated role of Tn phosphorylation crosstalk on cardiac contractile regulation.

The rate-limiting step of cardiac muscle relaxation has been proposed to reside in the myofilament. Both the rates of cross-bridge detachment and Ca(2+) dissociation from troponin C (TnC) have been hypothesized to rate-limit myofilament inactivation. In this study we used a fluorescent TnC to measure both the rate of Ca(2+) dissociation from TnC and the rate of cross-bridge detachment from several different species of ventricular myofibrils. The fluorescently labeled TnC was sensitive to both Ca(2+) dissociation and cross-bridge detachment at low Ca(2+) (presence of EGTA), allowing for a direct comparison between the two proposed rates of myofilament inactivation. Unlike Ca(2+) dissociation from TnC, cross-bridge detachment varied in myofibrils from different species and was rate-limited by ADP release. At subphysiological temperatures (20 °C), the rate of Ca(2+) dissociation from TnC was faster than the rate of cross-bridge detachment in the presence of ADP. These results support the hypothesis that cross-bridge detachment rate-limits relaxation. However, Ca(2+) dissociation from TnC was not as temperature-sensitive as cross-bridge detachment. At a near physiological temperature (35 °C) and ADP, the rate of cross-bridge detachment may actually be faster than the rate of Ca(2+) dissociation. This provides evidence that there may not be a simple, single rate-limiting step of myofilament inactivation.

Troponin C (TnC) is responsible for Ca2+-dependent activation and inactivation of the thin filament during contraction and relaxation of the heart. To study the Ca2+ binding properties of TnC, we designed a human cardiac TnC (C35S T53C C84S) ± the L48Q mutation. L48Q TnC has an increased Ca2+ sensitivity and decreased rate of Ca2+ dissocation as previously shown in multiple biochemical systems. We exchanged IANBD labeled control or L48Q Tn into rabbit ventricular myofibrils and used a stopped-flow apparatus to measure the fluorescence change related to Ca2+ dissociation. Unlike the mono-phasic Ca2+ dissociation curve of control myofibrils (25.3/s ± 0.7), Ca2+ dissociation from L48Q TnC myofibrils was biphasic, with rates of 17.3 ± 0.5/s and 4.73 ± 0.07/s, at 15oC. The apparent Ca2+ affinities of the fast and slow phases for L48Q TnC corresponded to the steady-state Ca2+ binding affinities of control and L48Q myofibrils, respectively. We hypothesized that the biphasic rate of L48Q Tn exchanged myofibrils results from the influence of the un-exchanged endogenous Tn. Consistent with this hypothesis, reconstituted thin filaments containing 100% L48Q Tn had a mono-phasic Ca2+ dissociation curve. Whereas, Ca2+ dissociation from reconstituted thin filaments containing IANBD labeled L48Q Tn and unlabeled control Tn lead to a biphasic Ca2+ dissociation rate, confirming the crosstalk between Tn subunits. Interestingly, the biphasic characteristic of L48Q TnC was absent when incorporated into diseased human ventricular myofibrils, possibly due to an altered thin filament state. Fluorescently labeled L48Q TnC provides insight into the interactions occurring between neighboring Tn subunits. Thus, L48Q TnC may be a useful tool to probe myofilament protein interactions along the thin filament.

Cardiac contraction relies upon the proper expression, trafficking, and retention of integral membrane proteins such as ion channels, transporters, and hormone receptors. These proteins govern cardiac contraction and short and long term adaptations to physiological and pathophysiological stimuli. The profile of expressed proteins is dynamic, and it is regulated to assure the proper response to stress. Yet, despite its obvious importance, little is known regarding even the identity (or the function) of the intracellular molecular pathways underpinning the trafficking and targeting of integral membrane proteins in the context of the native heart. The goal of this work is identify new molecular players that regulate protein targeting and trafficking in the heart. Eps 15 homology domain-containing (EHD) gene products (EHD1-4) are intracellular proteins that are key regulators of endosomal trafficking, lipid homeostasis, membrane protein recycling and trafficking. Previously uncharacterized in the heart, we recently presented evidence which demonstrated that this protein family likely plays indispensable roles in protein trafficking in cardiac muscle. Notably, an essential role for one of these proteins, EHD3, in the membrane trafficking of the Na/Ca exchanger (NCX) in heart was uncovered. The goal of this research program is for the first time to directly test the role of these proteins in cardiac structural and electrical activity using cutting-edge in vivo models of EHD deficiency. We show that EHD3 deficiency in the heart leads to: 1) abnormal cardiac structure at baseline; 2) irregular action potential morphology, heart rhythm, and conduction; 3) depressed β-adrenergic responsiveness; 4) dysfunctional NCX and LTCC trafficking and function; and 5) dysregulated ankyrin-B expression and trafficking. These data strongly support a role for EHD3 in membrane protein trafficking and regulation within the context of the native heart.

The rate of cardiac muscle relaxation is dynamically modulated. We hypothesize that the rate of Ca2+ dissociation from the thin filament is at least one of the factors that can strongly influence the rate of relaxation. Thus, we would expect the various physiological and pathological stimuli that affect the kinetics of cardiac muscle relaxation to likewise affect the rate of Ca2+ dissociation from the thin filament. In this study, we investigated various modulators of the Ca2+ exchange kinetics in the physiologically relevant biochemical model systems of reconstituted thin filaments and rabbit ventricular myofibrils such as: 1) PKA phosphorylation of TnI, 2) ischemia-reperfusion associated truncation of TnI, 3) familial cardiomyopathy related mutations of TnI and TnT, 4) the calcium sensitizing compound bepridil, 5) rationally engineered TnC mutations, and 6) tropomyosin isoforms. Consistent with the effects of PKA on accelerating relaxation, the rate of Ca2+ dissociation from the thin filament was accelerated by TnI mutations (S23D,S24D) mimicking PKA phosphorylation. Additionally, the rate of Ca2+ dissociation was slowed by truncation of TnI (residues 1-192), consistent with ischemia-reperfusion slowing the rate of cardiac relaxation. The hypertrophic and restrictive cardiomyopathy mutations (TnIS166F and TnIR192H) slowed the rate of Ca2+ dissociation from the thin filament, whereas the dilated cardiomyopathy mutations (TnTR141W and TnTdeltaK210) accelerated the rate of Ca2+ dissociation from the thin filament. The Ca2+ sensitizing compound bepridil had no effect on the rate of Ca2+ dissociation in either thin filaments or myofibrils, but engineered mutations of TnC can accelerate or slow the rate. Finally, the alpha and beta isoforms of tropomyosin slowed and accelerated the rate of Ca2+ dissociation from the thin filament, respectively. Thus, multiple factors can modulate the rate of Ca2+ dissociation from the thin filament.

Alteration of the Ca2+ binding properties of the thin filament has been implicated as the initiating factor for numerous cardiomyopathies. To directly test if changes in the Ca2+ sensitivity of TnC can lead to altered heart function and a diseased phenotype we utilized specifically engineered cardiac TnCs with modified Ca sensitivities. The specifically designed TnCs allowed us to test whether a direct increase (L48Q TnC) or decrease (D73N TnC) in Ca2+ sensitivity of the myofilament would lead to a hypertrophic or dilated cardiomyopathy, respectively. We used adeno-associated virus serotype 9 (AAV-9) containing either Control TnC, D73N TnC, or L48Q TnC to target the heart. AAV-9 containing GFP or mCherry was used to verify that the AAV-9 was able to infect the heart. The Ca2+ desensitized D73N TnC recapitulated a dilated cardiomyopathy. The hearts presented with dilated ventricles and had enlarged isolated cardiomyocytes that displayed decreased contractility. In addition, nearly half of the D73N mice died suddenly by 8 weeks of age and demonstrated altered electrical properties via ECG - a common feature of human dilated cardiomyopathies. On the other hand, AAV-9 containing the Ca2+ sensitized L48Q TnC did not recapitulate the restrictive or hypertrophic cardiomyopathy phenotypes commonly associated with increased myofilament Ca2+ sensitivity. However, consistent with the enhanced Ca2+ binding abilities of the L48Q TnC, the isolated L48Q TnC cardiomyocytes showed increased contractility, which was recapitulated in vivo via pressure-volume analysis. In summary, the results showed that an alteration in the Ca2+ sensitivity of the myofilament can, but does not always, lead to a diseased heart. In fact these engineered TnCs may be used as a treatment strategy against various cardiac diseases such as myocardial infarction.

It has been proposed that the rate limiting step of cardiac muscle relaxation resides in the myofilaments. The primary mechanism is thought to be the rate of cross-bridge detachment (strongly governed by ADP) since it is generally assumed to be substantially slower than the rate at which the thin filament inactivates (governed by Ca2+ dissociation from TnC). This stems from the fact that the rate of Ca2+ dissociation from isolated TnC is orders of magnitude faster than the rate of relaxation. However, TnC does not function in isolation but as an integral component of the myofilament contractile system. Furthermore, the Ca2+ binding properties of TnC can be drastically affected by the other thin filament proteins and by myosin binding to actin. Thus, we wanted to determine the Ca2+ dissociation rate from TnC in cardiac myofibrils during different cross-bridge states. To achieve this goal, rabbit ventricular myofibrils were exchanged with human cardiac troponin containing a TnC (C35S, C84S, T53C) fluorescently labeled with IANBD. Unexpectedly, via the change in TnC fluorescence, not only could we observe the rate of Ca2+ dissociation from TnC in the myofibrils, but also what we think is the rate of cross-bridge detachment. At 15oC and in the presence of ADP, the cross-bridge detachment rate was ∼7/s, three times slower than the rate of Ca2+ dissociation from TnC (∼21/s). However, at near physiological temperature (35oC) the two rates were very similar (∼60/s). Based on the temperature dependence of the rates, at temperatures below 25oC, cross-bridge detachment may very well be rate limiting for relaxation, but at higher temperatures both rates may be able to modulate the rate of relaxation.

The molecular events required for thin filament activation and deactivation in cardiac muscle are generally thought to be in rapid equilibrium. Stopped-flow measurements obtained with reconstituted thin filaments suggest that the rate of Ca2+ dissociation from cardiac TnC can be modulated by myosin S1 binding to actin and is not always rapid, but approaches the rate of mechanical relaxation at 15oC. To further probe the effects that myosin contributes to thin filament deactivation, we have utilized rabbit ventricular myofibrils as a more physiological biochemical system to examine the kinetics of Ca2+ dissociation from TnC. Incorporating a fluorescent TnC (C35S, C84S, T53C) labeled with IANBD into the myofibrils allows us to follow both the rates of Ca2+ dissociation from TnC, as well as what we think is cross-bridge (CB) detachment, through a change in IANBD fluorescence from TnC. However, CB detachment is only reported by the wild type TnC construct when there is no Ca2+ on the thin filament, while in the presence of ATP, Ca2+ dissociation is no longer observed. CB detachment can be observed in the presence of Ca2+ when Ca2+ binding to TnC is inactivated by D65A TnC. Additionally, the rate of Ca2+ dissociation from TnC can be observed when CBs are detached (presence of ATP) by the Ca2+ sensitizing mutations V44Q or L48Q TnC, but not when Ca2+ binding is desensitized by D73N TnC. Thus, weak and strong binding states of the thick filament differentially influence those of the thin filament within the cardiac myofibril, and are not necessarily in rapid equilibrium.

The troponin (Tn) complex is a critical regulatory and central integrative hub for post-translational modifications (PTM) within the thin filament. There are a number of Tn phosphorylations; however, β-adrenergic-induced protein kinase A (PKA) phosphorylation of cardiac troponin I (TnI) acts as the major sarcomeric modulator of function. Traditionally, individual phosphorylation sites have been studied, yet simultaneous phosphorylation of multiple sites occurs in the heart. To fully understand the molecular mechanisms underlying cardiac muscle regulation, Tn phosphorylation must be studied as integrated events. As an initial step towards understanding Tn crosstalk, we employed a two-model phosphomimetic approach using cardiac human Tn. We studied AMP-activated protein kinase (AMPK) TnI phosphorylation (TnI-S150D) and protein kinase C (PKC) cardiac troponin T (TnT) phosphorylation (TnT-T284E) to investigate the influence of these modifications on the effects exerted by TnI PKA phosphorylation (TnI-S23/24D). The combinatorial effects of these phosphorylation events on TnI-S23/24D was determined by measuring troponin C calcium binding properties in the reconstituted thin filament. Calcium dissociated from TnI-S23/24D thin filaments at 368/s compared to 103/s for wild-type (WT). Results demonstrate that TnI-S150D alone decreases calcium dissociation by 50%, while TnI-S150D integration with TnI-S23/24D blunted TnI-S23/24D kinetics by 15%, demonstrating the intramolecular crosstalk of TnI-S150D to alter the effects of TnI PKA phosphorylation. On the other hand, TnT-T284E calcium dissociation was identical to WT, but when present with TnI-S23/24D calcium dissociation was enhanced 136% compared to TnI-S23/24D alone. This demonstrates an intermolecular influence of TnT phosphorylation on TnI PKA effects. Overall, these findings demonstrate the combinatorial effects of Tn site-specific phosphorylation by AMPK and PKC crosstalk to affect the function of TnI PKA-induced phosphorylation through both intra- and inter- molecular mechanisms. These data further highlight the importance of understanding the role of integrated Tn PTM crosstalk on cardiac contractile regulation.

The FHC-related cTnC mutation, L29Q, is located at the dysfunctional binding site I of cTnC and interacts with the N-terminus of cardiac troponin I (cTnI) during muscle contraction. Recent studies suggest that the L29Q cTnC mutation affects Ca2+ activation and the transduction of PKA-dependent phosphorylation (S23/24) cTnI effect on cTnC, but the results are contradictory. Therefore, to investigate whether the L29Q cTnC mutation abrogates the effects of PKA-dependent phosphorylation of cTnI of the Ca2+ sensitivity of cTnC, we examined the Ca2+ binding properties in multiple biochemical systems of increasing complexity. Mouse cTn was reconstituted with phosphomimetic cTnI (S23/24D), cTnT, and IAANS-labeled control TnC (T53C, C84S, C35S) ± the L29Q mutation. Steady-state Ca2+ binding and Ca2+ dissociation rates were measured in the isolated cTn as well as the reconstituted thin filaments. There was no significant difference between steady-state Ca binding for the control cTn (0.98±0.07μM) and L29Q cTn (0.99±0.08μM), nor a difference in the Ca2+ dissociation kinetics of reconstituted thin filaments, control (300±10/s) and L29Q (280±10/s). However, L29Q cTnC in skinned cardiac myocytes increased Ca sensitivity by 1.2 fold and reduced the decrease in Ca2+ sensitivity caused by phosphomimetic cTnI by ∼ 45%, as well as the enhanced length dependent Ca2+ sensitivity. These results suggest that the Ca2+ association rate, rather than the dissociation rate was affected by L29Q in the reconstituted thin filament. In conclusion, the cTnC L29Q mutation increased Ca2+ sensitivity of force generation and exacerbated the functional effects of TnI phosphorylation at S23/24 in skinned cardiac myocytes, but did not alter changes in the Ca2+ dissociation rate in the reconstituted thin filament caused by phosphomimetic cTnI.