The renin-angiotensin (Ang) system regulates multiple physiological functions through Ang II type 1 and type 2 receptors. Prior studies suggest an intracellular pool of Ang II that may be released in an autocrine manner upon stretch to activate surface membrane Ang receptors. Alternatively, an intracellular renin-Ang system has been proposed, with a primary focus on nuclear Ang receptors. A mitochondrial Ang system has not been previously described. Here we report that functional Ang II type 2 receptors are present on mitochondrial inner membranes and are colocalized with endogenous Ang. We demonstrate that activation of the mitochondrial Ang system is coupled to mitochondrial nitric oxide production and can modulate respiration. In addition, we present evidence of age-related changes in mitochondrial Ang receptor expression, i.e., increased mitochondrial Ang II type 1 receptor and decreased type 2 receptor density that is reversed by chronic treatment with the Ang II type 1 receptor blocker losartan. The presence of a functional Ang system in human mitochondria provides a foundation for understanding the interaction between mitochondria and chronic disease states and reveals potential therapeutic targets for optimizing mitochondrial function and decreasing chronic disease burden with aging.

Rationale High-myofilament Ca2+-sensitivity has been proposed as trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) based on in vitro and transgenic mice studies. However, myofilament Ca2+-sensitivity depends on protein phosphorylation and muscle length, and at present, data in human are scarce. Objective To investigate whether high-myofilament Ca2+-sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick- and thin-filament proteins. Methods and Results Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca2+-sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA)-targets compared with donors. After exogenous PKA treatment, myofilament Ca2+-sensitivity was either similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations, but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values. Conclusions High-myofilament Ca2+-sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA-targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via post-translational modifications other than PKA-hypophosphorylation or altered protein–protein interactions, and represents a common pathomechanism in HCM.

Nitrovasodilators such as nitroglycerine, via production of nitric oxide and an increase in [cGMP], can induce arterial smooth muscle relaxation without proportional reduction in myosin light chain (MLC) phosphorylation or myoplasmic [Ca2+]. These findings suggest that regulatory systems, other than MLC phosphorylation and Ca2+, partially mediate nitroglycerine‐induced relaxation. In swine carotid artery, we found that a membrane‐permeant cGMP analogue induced relaxation without MLC dephosphorylation, suggesting that cGMP mediated the relaxation. Nitroglycerine‐induced relaxation was associated with a reduction in O2 consumption, suggesting that the interaction between phosphorylated myosin and the thin filament was inhibited. Nitroglycerine‐induced relaxation was associated with a 10‐fold increase in the phosphorylation of a protein on Ser16. We identified this protein as heat shock protein 20 (HSP20), a member of a family of proteins known to bind to thin filaments. When homogenates of nitroglycerine‐relaxed tissues were centrifuged at 6000 g, phosphorylated HSP20 preferentially sedimented in the pellet, suggesting that phosphorylation of HSP20 may increase its affinity for the thin filament. We noted that a domain of HSP20 is partially homologous to the ‘minimum inhibitory sequence’ of skeletal troponin I. The peptide HSP20110‐121, which contains this domain, bound to actin‐containing filaments only in the presence of tropomyosin, a characteristic of troponin I. High concentrations of HSP20110‐121 abolished Ca2+‐activated force in skinned swine carotid artery. HSP20110‐121 also partially decreased actin‐activated myosin S1 ATPase activity. These data suggest that cGMP‐mediated phosphorylation of HSP20 on Ser16 may have a role in smooth muscle relaxation without MLC dephosphorylation. HSP20 contains an actin‐binding sequence at amino acid residues 110–121 that inhibited force production in skinned carotid artery. We hypothesize that phosphorylation of HSP20 regulates force independent of MLC phosphorylation via binding of HSP20 to thin filaments and inhibition of cross‐bridge cycling.

Rationale Activation of the mitochondrial ATP-sensitive potassium channel (mitoKATP) has been implicated in the mechanism of cardiac ischemic preconditioning, yet its molecular composition is unknown. Objective To use an unbiased proteomic analysis of the mitochondrial inner membrane to identify the mitochondrial K+ channel underlying mitoKATP. Methods and Results Mass spectrometric analysis was used to identify KCNJ1(ROMK) in purified bovine heart mitochondrial inner membrane and confirmed that ROMK mRNA is present in neonatal rat ventricular myocytes and adult hearts. ROMK2, a short form of the channel, is shown to contain an N-terminal mitochondrial targeting signal and a full length epitope-tagged ROMK2 colocalizes with mitochondrial ATP synthase β. The high-affinity ROMK toxin, tertiapin Q, inhibits mitoKATP activity in isolated mitochondria and in digitonin-permeabilized cells. Moreover, shRNA-mediated knockdown of ROMK inhibits the ATP-sensitive, diazoxide activated, component of mitochondrial thallium uptake. Finally, the heart-derived cell line, H9C2, is protected from cell death stimuli by stable ROMK2 overexpression, while knockdown of the native ROMK exacerbates cell death. Conclusions The findings support ROMK as the pore-forming subunit of the cytoprotective mitoKATP channel.

mROS drive both acute emergent events, such as electrical instability responsible for SCD, and those that mediate chronic HF remodeling, characterized by suppression or altered phosphorylation of metabolic, antioxidant, and ion transport protein networks. In vivo reduction of mROS prevents and reverses electrical instability, SCD, and HF. Our findings support the feasibility of targeting the mitochondria as a potential new therapy for HF and SCD while identifying new mROS-sensitive protein modifications.

ATP is required for normal cardiac contractile function, and it has long been hypothesized that reduced energy delivery contributes to the contractile dysfunction of heart failure (HF). Despite experimental and clinical HF data showing reduced metabolism through cardiac creatine kinase (CK), the major myocardial energy reserve and temporal ATP buffer, a causal relationship between reduced ATP-CK metabolism and contractile dysfunction in HF has never been demonstrated. Here, we generated mice conditionally overexpressing the myofibrillar isoform of CK (CK-M) to test the hypothesis that augmenting impaired CK-related energy metabolism improves contractile function in HF. CK-M overexpression significantly increased ATP flux through CK ex vivo and in vivo but did not alter contractile function in normal mice. It also led to significantly increased contractile function at baseline and during adrenergic stimulation and increased survival after thoracic aortic constriction (TAC) surgery-induced HF. Withdrawal of CK-M overexpression after TAC resulted in a significant decline in contractile function as compared with animals in which CK-M overexpression was maintained. These observations provide direct evidence that the failing heart is "energy starved" as it relates to CK. In addition, these data identify CK as a promising therapeutic target for preventing and treating HF and possibly diseases involving energy-dependent dysfunction in other organs with temporally varying energy demands.

Congenital long-or short-QT syndrome may lead to life-threatening ventricular tachycardia and sudden cardiac death. Apart from the rare disease-causing mutations, common genetic variants in CAPON, a neuronal nitric oxide synthase (NOS1) regulator, have recently been associated with QT interval variations in a human whole-genome association study. CAPON had been unsuspected of playing a role in cardiac repolarization; indeed, its physiological role in the heart (if any) is unknown. To define the biological effects of CAPON in the heart, we investigated endogenous CAPON protein expression and protein-protein interactions in the heart and performed electrophysiological studies in isolated ventricular myocytes with and without CAPON overexpression. We find that CAPON protein is expressed in the heart and interacts with NOS1 to accelerate cardiac repolarization by inhibition of L-type calcium channel. Our findings provide a rationale for the association of CAPON gene variants with extremes of the QT interval in human populations.NOS1 ͉ QT interval ͉ cardiac electrophysiology R are disease-causing mutations leading to congenital long-or short-QT syndrome are well recognized, but there is little insight into genetic sources of QT interval variation in normal populations. A whole-genome association approach has recently implicated common genetic variants in CAPON as contributing to QT interval differences in a community-based German population (1). This association has since been confirmed in other populations (2, 3). The genetic findings challenge our current understanding of QT physiology. CAPON, first identified in rat brain neurons (4), is a highly conserved protein (Ϸ92% conceptual amino acid sequence identity between rat and human) with an N-terminal phosphotyrosine-binding (PTB) domain and a C-terminal PDZ-binding [postsynaptic density-95 (PSD95)/drosophila discs large/zona occludens-1] domain (4-6). In brain, CAPON competes with PSD95 for the binding of neuronal nitric oxide synthase (NOS1) through the interaction of its C terminus with the PDZ domain of NOS1 (4), thus uncoupling the NMDA-NOS1-NO-mediated signaling pathways. CAPON is also an adaptor protein of NOS1, capable of directing NOS1 to specific target proteins (5, 6). Nowhere, however, has CAPON been suspected of playing a role in cardiac physiology.Both NOS1 and endothelial NOS (NOS3) are constitutively expressed in cardiomyocytes (7). NOS1 in the sarcolemma has been proposed to interact with Na ϩ -K ϩ ATPase (8) and with the plasma membrane Ca 2ϩ /calmodulin-dependent Ca 2ϩ ATPase (PMCA) through the interaction of PDZ domain of NOS1 and the C terminus of PMCA4b isoform (9). In the sarcoplasmic reticulum (SR), NOS1 is structurally associated with ryanodine receptor 2 (RyR2) (10) and cardiac SR Ca 2ϩ ATPase (SERCA2) (11) to regulate intracellular calcium cycling and excitation-contraction coupling. Conditional transgenic overexpression of NOS1 in heart leads to additional association of NOS1 and the sarcolemmal L-type calcium channel and thus suppresses...

Abnormal smooth muscle contraction may contribute to diseases such as asthma and hypertension. Alterations to myosin light chain kinase or phosphatase change the phosphorylation level of the 20-kDa myosin regulatory light chain (MRLC), increasing Ca 2؉ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160 ROCK ) can induce Ca 2؉-independent contraction of Triton-skinned smooth muscle by phosphorylating MRLC and/or myosin light chain phosphatase. We show that another Rho family GTPase-dependent kinase, p21-activated protein kinase (PAK), induces Triton-skinned smooth muscle contracts independently of calcium to 62 ؎ 12% (n ‫؍‬ 10) of the value observed in presence of calcium. Remarkably, PAK and ROK use different molecular mechanisms to achieve the Ca 2؉ -independent contraction. Like ROK and myosin light chain kinase, PAK phosphorylates MRLC at serine 19 in vitro. However, PAK-induced contraction correlates with enhanced phosphorylation of caldesmon and desmin but not MRLC. The level of MRLC phosphorylation remains similar to that in relaxed muscle fibers (absence of GST-mPAK3 and calcium) even as the force induced by GST-mPAK3 increases from 26 to 70%. Thus, PAK uncouples force generation from MRLC phosphorylation. These data support a model of PAK-induced contraction in which myosin phosphorylation is at least complemented through regulation of thin filament proteins. Because ROK and PAK homologues are present in smooth muscle, they may work in parallel to regulate smooth muscle contraction.