Background Voltage-gated Na+ channels (Nav) are essential for myocyte membrane excitability and cardiac function. Nav current (INa) 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 INa (INa,L) persists due to incomplete/failed inactivation of a subpopulation of channels. Notably, INa,L is directly linked with both congenital and acquired disease states. The multifunctional Ca2+/calmodulin-dependent kinase II (CaMKII) has been identified as an important activator of INa,L in disease. Several potential CaMKII phosphorylation sites have been discovered, including Ser571 in the Nav1.5 DI-DII linker, but the molecular mechanism underlying CaMKII-dependent regulation of INa,L in vivo remains unknown. Methods and Results To determine the in vivo role of Ser571, two Scn5a knock-in mouse models were generated expressing either: 1) Nav1.5 with a phosphomimetic mutation at Ser571 (S571E), or 2) Nav1.5 with the phosphorylation site ablated (S571A). Electrophysiology studies revealed that Ser571 regulates INa,L but not other channel properties previously linked to CaMKII. Ser571-mediated increases in INa,L promote abnormal repolarization and intracellular Ca2+ 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 INa,L. Relevant for improved rational design of potential therapies, our findings demonstrate that Ser571-dependent regulation of Nav1.5 specifically tunes INa,L without altering critical physiological components of the current.

Background: Myofilament protein phosphorylation is central to cardiac muscle contractile regulation. Results: AMP-activated protein kinase (AMPK) phosphorylates troponin I (TnI) to increase cardiac contraction and blunt the effects of TnI protein kinase A phosphorylation. Conclusion: AMPK myofilament signaling represents a novel mechanism to regulate cardiac contraction. Significance: AMPK links metabolics to TnI regulation of cardiac muscle function and adrenergic regulation.

Rationale Nav1.5 (SCN5A) is the primary cardiac voltage-gated Nav channel. Nav1.5 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 Nav1.5 regulation are linked with malignant arrhythmias associated with human heart failure. Consequently, therapies to target select Nav1.5 properties have remained at the forefront of cardiovascular medicine. However, despite years of investigation, the fundamental pathways governing Nav1.5 membrane targeting, assembly, and regulation are still largely undefined. Objective Define the in vivo mechanisms underlying Nav1.5 membrane regulation. Methods and Results Here, we define the molecular basis of a Nav channel regulatory platform in heart. Using new cardiac-selective ankyrin-G−/− mice (cKO), we report that ankyrin-G targets Nav1.5, and its regulatory protein, calcium/calmodulin-dependent kinase II (CaMKII) to the intercalated disc. Mechanistically, βIV-spectrin is requisite for ankyrin-dependent targeting of CaMKIIδ, however βIV-spectrin is not essential for ankyrin-G expression. Ankyrin-G cKO myocytes display decreased Nav1.5 expression/membrane localization, and reduced INa associated with pronounced bradycardia, conduction abnormalities, and ventricular arrhythmia in response to Nav channel antagonists. Moreover, we report that ankyrin-G links Nav channels with broader intercalated disc signaling/structural nodes, as ankyrin-G loss results in reorganization of plakophilin-2 and lethal arrhythmias in response to beta-adrenergic stimulation. Conclusions Our findings provide the first in vivo data for the molecular pathway required for intercalated disc Nav1.5 targeting/regulation in heart. Further, these new data identify the basis of an in vivo cellular platform critical for membrane recruitment and regulation of Nav1.5.

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 calcium dependent interactions between troponin C (TnC) and other thin and thick filament proteins play a key role in 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) calcium binding and exchange with TnC in increasingly complex biochemical systems and 2) 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 inter-molecular 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 calcium binding to TnC in increasingly complex biochemical systemsThe processes of cardiac muscle contraction and relaxation can be regulated by multiple physiological and patho-physiological stimuli. It is clear that protein alterations associated with heart disease, isoform switching and post translational modifications can affect both the Ca 2+ sensitivity of muscle force generation and relaxation kinetics (for review, see (1-4)). Since cardiac troponin C (TnC) 1 is the Ca 2+ sensor responsible for initiating the contraction / relaxation cycle (for review, see (5,6)), a potentially important mechanism to alter cardiac † This research was funded by NIH grants 5R00HL087462 (to S.B.T) and 5R01HL073828 (to D.R.S.); by Predoctoral Fellowship Award from the American Heart Association (to B. L.) and by National Scientist Development Award from the American Heart Association (to J.P.D).*Address correspondence to: Jonathan P. Davis, Department of Physiology and Cell Biology, The Ohio State University, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210, Tel. 614-247-2559; Fax. 614-292-4888; davis.812@osu.edu. ‡ These two authors contributed equally to the manuscript NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 March 9. Published in final edited form as:Biochemistry. 2010 March 9; 49(9): 1975-1984. doi:10.1021/bi901867s. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript muscle performance is through directly modifying the properties of TnC. As it has been difficult to find specific pharmacological modulators of TnC, we have taken a genetic approach to modify Ca 2+ binding and exchange with TnC.I...

Background: PP2A regulates cardiac excitability and physiology. Results: PP2A regulation in heart occurs through integrative transcriptional, translational, and post-translational control of three classes of subunits (17 genes) to control holoenzyme synthesis, localization, and maintenance; pathways are mechanistically altered in heart disease. Conclusion:Multiple mechanisms are present for acute and chronic regulation of specific PP2A populations. Significance: Results provide molecular insight into cardiac PP2A regulation.

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 with cardiac pathologies including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction in cardiac electrical 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 post-translational 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 electrical 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 that include the Na/Ca exchanger, RyR2, 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 electrical activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology.

The binding of Ca2+ 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 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 Ca2+ to TnC and impaired contraction. To determine the pathological relevance of simultaneous TnI phosphorylation 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 Ca2+ 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 Ca2+ 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-phosphorylation to maintain Ca2+ binding while accelerating Ca2+ dissociation represents the first post-translational modification of troponin by phosphorylation to both accelerate thin filament deactivation and maintain Ca2+ sensitive activation. These data suggest TnI Ser-150 phosphorylation attenuation of the pH-dependent decrease in Ca2+ 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.