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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.

Ischemia and reperfusion affect multiple elements of cardiomyocyte electrophysiology, especially within the mitochondria. We previously showed that in cardiac monolayers, upon reperfusion after coverslip-induced ischemia, mitochondrial inner membrane potential (ΔΨ) unstably oscillates between polarized and depolarized states, and ΔΨ instability corresponds with arrhythmias. Here, through confocal microscopy of compartment-specific molecular probes, we investigate the mechanisms underlying the post-ischemic ΔΨ oscillations, focusing on the role of Ca

[Figure: see text].

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.

Mitochondrial dysfunction contributes to heart failure. Our recent work revealed that genes in mitochondrial biogenesis and quality control, such as the peroxisome proliferator activated receptor gamma coactivator-1α(PGC-1α) and mitochondrial transcription factor A(TFAM), are downregulated in a rodent model of heart failure. Here, we apply CRISPR/Cas9 technology as a transcriptional activator to target sequences upstream of the start site to promote transcription of PGC-1α or TFAM. Since cardiomyocytes are difficult to transduce, we developed an adenoviral gene delivery system for expression of the CRISPR/Cas9 activating complex and a targeting gRNA to modulate expression of any gene of interest in differentiated cardiomyocytes, with our initial focus on activating the metabolic gene program. We use nuclease-dead Cas9 (ndCas9) fused to an activation domain VP64 and guide RNAs for activation of target genes. Proof-of-principle evidence was obtained by activating a cyclic AMP response element (CRE)-driven luciferase reporter gene and activation of endogenous CRE-regulable genes (PCK1 and PGC-1α). Next, we constructed ndCas9-gRNA adenoviral vectors to target PGC-1α and TFAM in the rat genome to determine if transcription of these genes can be increased. Functional effects of viral gene transduction in H9C2 cells and neonatal rat ventricular myocytes are assessed by determining mitochondrial DNA copy number (mtDNA)/genomic DNA ratio, and mtDNA quantity by picogreen labeling. Results: We have successfully upregulated the luciferase gene and endogenous CRE-regulated gene PCK1 in the same reporter cell line with ndCas9-VP64, as evidenced by significant increases in mRNA and protein expression levels. We have constructed and delivered adenoviral vectors with ndCas9 that are efficiently expressed by the host cells (NRVMs and H9c2 cells) and showed increased expression of PGC1a and TFAM in H9c2 cells. Importantly, an increase in mtDNA copy number was evoked by TFAM activation. For the first time, we show that the CRISPR/ndCas9 system can be used as a powerful tool for modulating transcription of native genes involved in mitochondrial biogenesis in cardiac cells, opening the door to developing novel therapeutic approaches for heart failure

Oxidative stress arises from an imbalance in the production and scavenging rates of reactive oxygen species (ROS) and is a key factor in the pathophysiology of cardiovascular disease and aging. The presence of parallel pathways and multiple intracellular compartments, each having its own ROS sources and antioxidant enzymes, complicates the determination of the most important regulatory nodes of the redox network. Here we quantified ROS dynamics within specific intracellular compartments in the cytosol and mitochondria and determined which scavenging enzymes exert the most control over antioxidant fluxes in H9c2 cardiac myoblasts. We used novel targeted viral gene transfer vectors expressing redox-sensitive GFP fused to sensor domains to measure H2O2 or oxidized glutathione. Using genetic manipulation in heart-derived H9c2 cells, we explored the contribution of specific antioxidant enzymes to ROS scavenging and glutathione redox potential within each intracellular compartment. Our findings reveal that antioxidant flux is strongly dependent on mitochondrial substrate catabolism, with availability of NADPH as a major rate-controlling step. Moreover, ROS scavenging by mitochondria significantly contributes to cytoplasmic ROS handling. The findings provide fundamental information about the control of ROS scavenging by the redox network and suggest novel interventions for circumventing oxidative stress in cardiac cells.

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.

Insulin can alter myocardial contractility, in part through an effect on the cardiac sarcolemmal Na(+)/Ca(2+) exchanger (NCX), but little is known about its mechanism of action. The large cytoplasmic domain (f-loop) of NCX is required for regulation by various intracellular factors, and we have shown previously that residues 562-679 are determinants of NCX inhibition by exchanger inhibitory peptide (XIP). Here we show that the same f-loop deletion eliminates the enhancement of NCX current by insulin, and we examine the signal pathways involved in the insulin response. NCX current (I(NCX)) was measured in freshly isolated or cultured (up to 48 h) adult guinea pig myocytes and in myocytes expressing canine NCX1.1 with the 562-679 f-loop deletion (NCX-(Delta562-679)) via adenoviral gene transfer. I(NCX) was recorded by whole-cell patch clamp as the Ni(2+)-sensitive current at 37 degrees C with intracellular Ca(2+) buffered. Insulin (1 microm) increased I(NCX) (at +80 mV) by 110 and 83% in fresh and cultured myocytes, respectively, whereas in myocytes expressing NCX-(Delta562-679) the response was eliminated (with 100 microm XIP included to suppress any native guinea pig I(NCX)). The insulin effect on I(NCX) was not inhibited by wortmannin, a nitric-oxide synthase inhibitor, or disruption of caveolae but was blocked by chelerythrine, implicating protein kinase C, but not phosphatidylinositol-3-kinase, in the mechanism. The insulin effect was also not additive with phosphatidylinositol-4,5-bisphosphate-induced activation of I(NCX). The finding that the 562-670 f-loop domain is implicated in both XIP and receptor-mediated modulation of NCX highlights its important role in acute physiological or pathophysiological regulation of Ca(2+) balance in the heart.

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.

Insulin can alter myocardial contractility, in part through an effect on the cardiac sarcolemmal Na(+)/Ca(2+) exchanger (NCX), but little is known about its mechanism of action. The large cytoplasmic domain (f-loop) of NCX is required for regulation by various intracellular factors, and we have shown previously that residues 562-679 are determinants of NCX inhibition by exchanger inhibitory peptide (XIP). Here we show that the same f-loop deletion eliminates the enhancement of NCX current by insulin, and we examine the signal pathways involved in the insulin response. NCX current (I(NCX)) was measured in freshly isolated or cultured (up to 48 h) adult guinea pig myocytes and in myocytes expressing canine NCX1.1 with the 562-679 f-loop deletion (NCX-(Delta562-679)) via adenoviral gene transfer. I(NCX) was recorded by whole-cell patch clamp as the Ni(2+)-sensitive current at 37 degrees C with intracellular Ca(2+) buffered. Insulin (1 microm) increased I(NCX) (at +80 mV) by 110 and 83% in fresh and cultured myocytes, respectively, whereas in myocytes expressing NCX-(Delta562-679) the response was eliminated (with 100 microm XIP included to suppress any native guinea pig I(NCX)). The insulin effect on I(NCX) was not inhibited by wortmannin, a nitric-oxide synthase inhibitor, or disruption of caveolae but was blocked by chelerythrine, implicating protein kinase C, but not phosphatidylinositol-3-kinase, in the mechanism. The insulin effect was also not additive with phosphatidylinositol-4,5-bisphosphate-induced activation of I(NCX). The finding that the 562-670 f-loop domain is implicated in both XIP and receptor-mediated modulation of NCX highlights its important role in acute physiological or pathophysiological regulation of Ca(2+) balance in the heart.

Ion channels in the mitochondrial inner membrane influence the response of the heart to ischemia and reperfusion - the opening of mitochondrial K + channels (mitoK Ca or mitoK ATP ) induce cardioprotection while the excessive activation of an inner membrane anion channel (IMAC) contributes to post-ischemic contractile and electrical dysfunction. Here we examined the effects of K Ca channel openers and inhibitors on mitochondrial volume and respiration in KCl-based medium in energized isolated guinea-pig heart mitochondria. Inhibition of IMAC with a mitochondrial benzodiazepine ligand was also assessed. Mitochondrial swelling and contraction were evoked by the sequential addition of 5mM glutamate (Glu) and 5mM malate (Mal), respectively. Both the swelling and contraction responses involved energy-dependent fluxes of K + and Cl − . The Glu-induced swelling response was suppressed by preincubation with either the K Ca channel toxin paxilline, or by the IMAC blocker 4′-chlorodiazepam (4-Cl-DZP). The K Ca opener NS1619, at concentrations 10μM. Under similar conditions, mitochondrial respiratory control (State3/State4 VO 2 ratio) was maintained at >15 for [NS1619] 0.5 for ADP. The findings indicate that mitochondrial matrix volume is modulated by mitoK Ca channel activity under normal conditions and in the presence of K Ca channel openers, while IMAC channels underlie compensatory anion movements in response to K + flux. Physiological regulation of mitochondrial volume by ion transport is in turn linked to altered respiratory flux, with important implications for cardioprotection and recovery of mitochondrial function in the post-ischemic heart.

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 alpha(1c) subunit of the cardiac L-type Ca(2+) channel, which contains the channel pore, voltage- and Ca(2+)-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca(2+) channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length alpha(1c) gene cassettes into the adenovirus backbone. Wild-type (alpha(1c-wt)) and mutant (alpha(1c-D-)) Ca(2+) channel adenoviruses were constructed. The alpha(1c-D-) contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both alpha(1c-wt) and alpha(1c-D-) expressed robustly in A549 cells (peak L-type Ca(2+) current (I(CaL)) at 0 mV: alpha(1c-wt) -9.94+/-1.00pA/pF, n=9; alpha(1c-D-) -10.30pA/pF, n=12). I(CaL) carried by alpha(1c-D-) was markedly less sensitive to nitrendipine (IC(50) 17.1 microM) than alpha(1c-wt) (IC(50) 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 microM nitrendipine blocked only 51+/-5% (n=9) of I(CaL) in alpha(1c-D-)-expressing myocytes, in comparison to 86+/-8% (n=9) of I(CaL) in control myocytes. Moreover, in 20 microM nitrendipine, calcium transients could still be evoked in alpha(1c-D-)-transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca(2+) release. These alpha(1c) adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca(2+) channel regulation in the native myocyte background.

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.

Ion channels on the mitochondrial inner membrane influence cell function in specific ways that can be detrimental or beneficial to cell survival. At least one type of potassium (K + ) channel, the mitochondrial adenosine triphosphate–sensitive K + channel (mitoK ATP ), is an important effector of protection against necrotic and apoptotic cell injury after ischemia. Here another channel with properties similar to the surface membrane calcium-activated K + channel was found on the mitochondrial inner membrane (mitoK Ca ) of guinea pig ventricular cells. MitoK Ca significantly contributed to mitochondrial K + uptake of the myocyte, and an opener of mitoK Ca protected hearts against infarction.

Cyclic AMP is a major second messenger that inhibits the brush border Na+/H+ exchanger NHE3. We have previously shown that either of two related regulatory proteins, E3KARP or NHERF, is necessary for the cAMP-dependent inhibition of NHE3. In the present study, we characterized the interaction between NHE3 and E3KARP using in vitro binding assays. We found that NHE3 directly binds to E3KARP and that the entirety of the second PSD-95/Dlg/ZO-1 (PDZ) domain plus the carboxyl-terminal domain of E3KARP are required to bind NHE3. E3KARP binds an internal region within the NHE3 C-terminal cytoplasmic tail, defining a new mode of PDZ domain interaction. Analyses of cellular distribution of NHE3 and E3KARP expressed in PS120 fibroblasts show that NHE3 and E3KARP are co-localized on the plasma membrane, but not in a distinct juxtanuclear compartment in which NHE3 is predominantly expressed. The distributions of NHE3 and E3KARP were not affected by treatment with 8-bromo-cAMP. As shown earlier for the human homolog of NHERF, we also found that the cytoskeletal protein ezrin binds to the carboxyl-terminal domain of E3KARP. These results are consistent with the possibility that E3KARP and NHERF may function as scaffold proteins that bind to both NHE3 and ezrin. Since ezrin is a protein kinase A anchoring protein, we suggest that the scaffolding function of E3KARP binding to both ezrin and NHE3 localizes cAMP-dependent protein kinase in the vicinity of the cytoplasmic domain of NHE3, which is phosphorylated by elevated cAMP.

Background: Mitochondria produce reactive oxygen species (ROS) that are scavenged by local antioxidant enzymes. Glutathione (GSH) is key intermediate in many of these reactions and its availability determines the antioxidant capacity of mitochondria. Glutathione S-transferases (GSTs) are known to consume GSH during xenobiotic detoxification but their involvement in ROS scavenging is less clear. Gstκ1 was originally identified as a novel mitochondrial GST but its role there remains unknown.Objective: To examine whether loss of Gstκ1 affects the [GSSG/GSH] ratios in cytosol and mitochondria of the cardiac-derived cell line H9c2.Methods: Knockdown of Gstκ1 (Gstκ1-KD) in H9c2 cells was achieved by transfection with siRNA. Changes in [GSSG/GSH] were monitored using genetically-encoded ratiometric sensors that localize to cytosol or mitochondria. Global oxidative stress was induced with hydrogen peroxide (HP). Mitochondrially-targeted oxidative stress was induced by the superoxide generator di-methoxy-naphtho-quinone (DMNQ) and by antimycin-A (AA) which interacts specifically with mitochondrial complex III.Results: Treatment with HP elevatedcytosolic [GSSG/GSH] ratios equally in both control and Gstκ1-KD cells. Mitochondrial oxidative stress elicited by HP or DMNQ likewise increased mitochondrial [GSSG/GSH] and was unaffected by Gstκ1-KD. Inhibition of Complex III by AA decreased mitochondrial [GSSG/GSH] persistently in control cells. However, in Gstκ1-KD cells exposed to AA, the decrease was transient and was followed by a sustained increase in [GSSG/GSH].Conclusions: Here we examine for the first time the role of Gstκ1 in ROS scavenging in cardiac cells. While our data suggest that Gstκ1 does not participate in the general response against exogenous ROS, they indicate that low levels of this enzyme associate with increased superoxide production from mitochondrial complex III. Our findings may provide insights for experimental models of cardiac disease where Gstκ1 expression is downregulated.