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Rationale: In heart failure (HF), impaired sarcoplasmic reticulum (SR) Ca 2+ release and cytosolic Na + overload depress mitochondrial Ca 2+ (mCa 2+ ) signaling, resulting in a diminished ability to maintain matrix NAD(P)H redox potential, leading to increased oxidative stress when workload increases. Enhancing mCa 2+ can reverse this defect but could potentially increase the likelihood of mCa 2+ overload. Objective: To determine if moderate mCa 2+ uniporter (MCU) overexpression has beneficial or detrimental effects on the development of HF and incident arrythmias in a guinea pig model (ACi) of HF and sudden cardiac death. Methods and Results: In vivo viral gene transfer was used to increase MCU levels by ≈57% in ACi hearts. Left ventricular myocytes from hearts with MCU overexpression (ACi+MCU) displayed enhanced mCa 2+ uptake, decreased oxidative stress, and increased β-adrenergic- and frequency-dependent augmentation of Ca 2+ transients and contractions, compared with myocytes from ACi hearts. MCU overexpression decreased SR Ca 2+ leak in the ACi group and mitigated the elevated RyR (ryanodine receptor) disulfide crosslinks in HF. β-Adrenergic responses were blunted in isolated perfused ACi hearts, and these deficiencies were normalized in ACi+MCU hearts. To examine the in vivo effects of MCU overexpression, ACi hearts were transduced with the MCU virus 2 to 3 weeks after aortic constriction, at the onset of cardiac decompensation. Two weeks later, cardiac function worsened in the untreated ACi group (fractional shortening: 39±1% at 2 weeks and 32±1% at 4 weeks), whereas MCU overexpression significantly improved cardiac function (36±1% at 2 weeks and 42±2% at 4 weeks). MCU overexpression in the decompensating ACi heart also attenuated pulmonary edema and interstitial fibrosis and prevented triggered arrhythmias. Conclusions: Moderate MCU overexpression in failing hearts enhances contractility and responses to β-adrenergic stimulation in isolated myocytes and perfused hearts by inhibiting mitochondrial oxidative stress-induced SR Ca 2+ leak. MCU overexpression also reversed HF and inhibited ectopic ventricular arrhythmias.

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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: 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 : In cardiomyocytes from failing hearts, insufficient mitochondrial Ca 2+ accumulation secondary to cytoplasmic Na + overload decreases NAD(P)H/NAD(P) + redox potential and increases oxidative stress when workload increases. These effects are abolished by enhancing mitochondrial Ca 2+ with acute treatment with CGP-37157 (CGP), an inhibitor of the mitochondrial Na + /Ca 2+ exchanger. Objective : Our aim was to determine whether chronic CGP treatment mitigates contractile dysfunction and arrhythmias in an animal model of heart failure (HF) and sudden cardiac death (SCD). Methods and Results : Here, we describe a novel guinea pig HF/SCD model using aortic constriction combined with daily β-adrenergic receptor stimulation (ACi) and show that chronic CGP treatment (ACi plus CGP) attenuates cardiac hypertrophic remodeling, pulmonary edema, and interstitial fibrosis and prevents cardiac dysfunction and SCD. In the ACi group 4 weeks after pressure overload, fractional shortening and the rate of left ventricular pressure development decreased by 36% and 32%, respectively, compared with sham-operated controls; in contrast, cardiac function was completely preserved in the ACi plus CGP group. CGP treatment also significantly reduced the incidence of premature ventricular beats and prevented fatal episodes of ventricular fibrillation, but did not prevent QT prolongation. Without CGP treatment, mortality was 61% in the ACi group Conclusions : The findings demonstrate the critical role played by altered mitochondrial Ca 2+ dynamics in the development of HF and HF-associated SCD; moreover, they reveal a novel strategy for treating SCD and cardiac decompensation in HF.

β-Adrenoceptor stimulation robustly increases cardiac L-type Ca 2+ current ( I CaL ); yet the molecular mechanism of this effect is still not well understood. Previous reports have shown in vitro phosphorylation of a consensus protein kinase A site at serine 1928 on the carboxyl terminus of the α 1C subunit; however, the functional role of this site has not been investigated in cardiac myocytes. Here, we examine the effects of truncating the distal carboxyl terminus of the α 1C subunit at amino acid residue 1905 or mutating the putative protein kinase A site at serine 1928 to alanine in adult guinea pig myocytes, using novel dihydropyridine-insensitive α 1C adenoviruses, coexpressed with β 2 subunits. Expression of α 1C truncated at 1905 dramatically attenuated the increase of peak I CaL induced by isoproterenol. However, the point mutation S1928A did not significantly attenuate the β-adrenergic response. The findings indicate that the distal carboxyl-terminus of α 1C plays an important role in β-adrenergic upregulation of cardiac L-type Ca 2+ channels, but that phosphorylation of serine 1928 is not required for this effect.

The sarcolemmal Na + -Ca 2+ exchanger (NCX) is the main Ca 2+ extrusion mechanism in cardiac myocytes and is thus essential for the regulation of Ca 2+ homeostasis and contractile function. A cytosolic region (f-loop) of the protein mediates regulation of NCX function by intracellular factors including inhibition by exchanger inhibitory peptide (XIP), a 20 amino acid peptide matching the sequence of an autoinhibitory region involved in allosteric regulation of NCX by intracellular Na + , Ca 2+ , and phosphatidylinositol-4,5-biphosphate (PIP 2 ). Previous evidence indicates that the XIP interaction domain can be eliminated by large deletions of the f-loop that also remove activation of NCX by intracellular Ca 2+ . By whole-cell voltage clamping experiments, we demonstrate that deletion of residues 562–679, but not 440– 456, 498–510, or 680–685 of the f-loop selectively eliminates XIP-mediated inhibition of NCX expressed either heterologously (HEK293 and A549 cells) or in guinea pig cardiac myocytes. In contrast, by plotting I NCX against reverse-mode NCX-mediated Ca 2+ transients in myocytes, we demonstrate that Ca 2+ -dependent regulation of NCX is preserved in Δ562–679, but significantly reduced in the other three deletion mutants. The findings indicate that f-loop residues 562–679 may contain the regulatory site for endogenous XIP, but this site is distinct from the Ca 2+ -regulatory domains of the NCX. Because regulation of the NCX by Na + and PIP 2 involves the endogenous XIP region, the Δ562–679 mutant NCX may be a useful tool to investigate this regulation in the context of the whole cardiac myocyte.