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:: Despite increasing prevalence and incidence of heart failure (HF), therapeutic options remain limited. In early stages of HF, sudden cardiac death (SCD) from ventricular arrhythmias claims many lives. Reactive oxygen species (ROS) have been implicated in both arrhythmias and contractile dysfunction. However, little is known about how ROS in specific subcellular compartments contribute to HF or SCD pathophysiology. The role of ROS in chronic proteome remodeling has not been explored. Objective:: We will test the hypothesis that elevated mitochondrial ROS (mROS) is a principal source of oxidative stress in HF and in vivo reduction of mROS mitigates SCD. Methods and Results:: Using a unique guinea pig model of nonischemic HF that recapitulates important features of human HF, including prolonged QT interval and high incidence of spontaneous arrhythmic SCD, compartment-specific ROS sensors revealed increased mROS in resting and contracting left ventricular myocytes in failing hearts. Importantly, the mitochondrially targeted antioxidant (MitoTEMPO) normalized global cellular ROS. Further, in vivo MitoTEMPO treatment of HF animals prevented and reversed HF, eliminated SCD by decreasing dispersion of repolarization and ventricular arrhythmias, suppressed chronic HF-induced remodeling of the expression proteome, and prevented specific phosphoproteome alterations. Pathway analysis of mROS-sensitive networks indicated that increased mROS in HF disrupts the normal coupling between cytosolic signals and nuclear gene programs driving mitochondrial function, antioxidant enzymes, Ca ²⁺ handling, and action potential repolarization, suggesting new targets for therapeutic intervention. Conclusions:: 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.

Rationale:: High-myofilament Ca ²⁺ sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca ²⁺ sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce. Objective:: To investigate whether high myofilament Ca ²⁺ 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 Ca ²⁺ sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca ²⁺ sensitivity was similar ( MYBPC3 _mut , TPM1 _mut , sarcomere mutation-negative HCM), higher ( MYH7 _mut , TNNT2 _mut ), or even significantly lower ( TNNI3 _mut ) 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 TNNT2 _mut and TNNI3 _mut corrected length-dependent activation to donor values. Conclusions:: High-myofilament Ca ²⁺ 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 posttranslational modifications other than PKA hypophosphorylation or altered protein–protein interactions, and represents a common pathomechanism in HCM.

Rationale:: Activation of the mitochondrial ATP-sensitive potassium channel (mitoK _ATP ) 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 mitoK _ATP . Methods and Results:: Mass spectrometric analysis was used to identify KCNJ1(ROMK) in purified bovine heart mitochondrial inner membrane and ROMK mRNA was confirmed to be 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 mitoK _ATP activity in isolated mitochondria and in digitonin-permeabilized cells. Moreover, short hairpin RNA—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, whereas knockdown of the native ROMK exacerbates cell death. Conclusions:: The findings support ROMK as the pore-forming subunit of the cytoprotective mitoK _ATP channel.

Rationale:: Cardiac resynchronization therapy (CRT) is an effective clinical treatment for heart failure patients with conduction delay, impaired contraction, and energetics. Our recent studies have revealed that mitochondrial posttranslational modifications (PTM) may contribute to its benefits, motivating the present study of the oxidative regulation of mitochondrial ATP synthase. Objectives:: We tested whether CRT alteration of ATP synthase function is linked to cysteine (Cys) oxidative PTM (Ox-PTM) of specific ATP synthase subunits. Methods and Results:: Canine left ventricular myocardium was collected under conditions to preserve Ox-PTM from control, dyssynchronous heart failure (DHF), or hearts that had undergone CRT. In-gel ATPase activity showed that CRT increased ATPase activity by ≈2-fold ( P <0.05) over DHF, approaching control levels, and this effect was recapitulated with a reducing agent. ATP synthase function and 3 Ox-PTM: disulfide bond, S-glutathionylation and S-nitrosation were assessed. ATP synthase from DHF hearts contained 2 novel disulfide bonds, between ATP synthase α subunits themselves and between α and γ subunits, both of which were decreased in CRT hearts (4.38±0.13- and 4.23±0.36-fold, respectively, P <0.01). S-glutathionylation of ATP synthase α subunit occurred in DHF hearts and was decreased by CRT (1.56±0.16-fold, P <0.04). In contrast, S-nitrosation of ATP synthase α subunit in DHF hearts was lower than in CRT hearts (1.53±0.19-fold, P <0.05). All modifications occurred at ATP synthase α subunit Cys294 and Cys to Ser mutation indicated that this residue is critical for ATP synthase function. Conclusions:: A selective Cys in ATP synthase α subunit is targeted by multiple Ox-PTM suggesting that this Cys residue may act as a redox sensor modulating ATP synthase function.

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 Ca²⁺ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160^ROCK) can induce Ca²⁺-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 Ca²⁺-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.