Hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction.

Mitochondria alter their shape by undergoing cycles of fusion and fission. Changes in mitochondrial morphology impact on the cellular response to stress, and their interactions with other organelles such as the sarcoplasmic reticulum (SR). Inhibiting mitochondrial fission can protect the heart against acute ischemia/reperfusion (I/R) injury. However, the role of the mitochondrial fusion proteins, Mfn1 and Mfn2, in the response of the adult heart to acute I/R injury is not clear, and is investigated in this study. To determine the effect of combined Mfn1/Mfn2 ablation on the susceptibility to acute myocardial I/R injury, cardiac-specific ablation of both Mfn1 and Mfn2 (DKO) was initiated in mice aged 4-6 weeks, leading to knockout of both these proteins in 8-10-week-old animals. This resulted in fragmented mitochondria (electron microscopy), decreased mitochondrial respiratory function (respirometry), and impaired myocardial contractile function (echocardiography). In DKO mice subjected to in vivo regional myocardial ischemia (30 min) followed by 24 h reperfusion, myocardial infarct size (IS, expressed as a % of the area-at-risk) was reduced by 46% compared with wild-type (WT) hearts. In addition, mitochondria from DKO animals had decreased MPTP opening susceptibility (assessed by Ca(2+)-induced mitochondrial swelling), compared with WT hearts. Mfn2 is a key mediator of mitochondrial/SR tethering, and accordingly, the loss of Mfn2 in DKO hearts reduced the number of interactions measured between these organelles (quantified by proximal ligation assay), attenuated mitochondrial calcium overload (Rhod2 confocal microscopy), and decreased reactive oxygen species production (DCF confocal microscopy) in response to acute I/R injury. No differences in isolated mitochondrial ROS emissions (Amplex Red) were detected in response to Ca(2+) and Antimycin A, further implicating disruption of mitochondria/SR tethering as the protective mechanism. In summary, despite apparent mitochondrial dysfunction, hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction due to impaired mitochondria/SR tethering.

Mitochondrial G protein coupled receptor kinase 2 regulates proinflammatory responses in macrophages.

G-protein-coupled receptor kinase 2 (GRK2) levels are elevated in inflammation but its role is not clear yet. Here we show that GRK2 expression is dependent on NFκB transcriptional activity. In macrophages, LPS induces GRK2 accumulation in mitochondria increasing biogenesis. The overexpression of the carboxy-terminal domain of GRK2 (ßARK-ct), known to displace GRK2 from plasma membranes, induces earlier localization of GRK2 to mitochondria in response to LPS leading to increased mt-DNA transcription and reduced ROS production and cytokine expression. Our study shows the relevance of GRK2 subcellular localization in macrophage biology and its potential therapeutic properties in inflammation.

Reduced troponin I phosphorylation and increased Ca(2+)-dependent ATP-consumption in triton X-skinned fiber preparations from Galphaq overexpressor mice.

Overexpression of the Galphaq-protein has been shown to result in hypertrophic and dilated cardiomyopathy. This study investigated Ca(2+ )sensitivity of tension and myosin-ATPase activity in skinned fiber preparations of male and female wildtype (WT; n = 12) and transgenic mice with a cardiac specific overexpression of the Galphaq-protein (Galphaq-OE; n = 11). In addition, the phosphorylation status of troponin I was measured. Ca(2+) sensitivity of tension was increased in Galphaq-OE with a significant reduction in the half-maximum Ca(2+) concentration (EC(50)) compared to WT. Similarly, Ca(2+) sensitivity of myosin ATPase activity was increased in Galphaq-OE when comparing Galphaq-OE to WT. Maximum Ca(2+)-dependent tension and ATPase activity were both enhanced in Galphaq-OE compared to WT littermates. Phosphorylation of troponin I was significantly reduced in Galphaq-OE compared to WT. In the above experiments, no gender specific differences were observed in either Gaq-OE or in WT. We conclude that, in mice, increased expression of the Galphaq-protein induces alterations of myofibrillar function and energy consumption, which are also characteristics of human heart failure. This may result from a decreased phosphorylation of troponin I in Galphaq-OE.

Cardiac reanimation: targeting cardiomyocyte death by BNIP3 and NIX/BNIP3L.

Programmed cardiac myocyte death contributes to pathological ventricular remodeling and the progression of myocardial infarction or pressure overload hypertrophy to dilated cardiomyopathy. Recent work has identified importance of stress-mediated transcriptional induction of BNIP3 (BCL2 and 19-kDa interacting protein-3) and NIX/BNIP3L in cardiac remodeling. Here, the regulatory mechanisms for these two factors in the heart and their effects on programmed cardiomyocyte death are reviewed, with a focus on information derived from studies using mouse models of cardiac BNIP3 and NIX/BNIP3L overexpression and gene ablation.

Increased myocardial Rab GTPase expression: a consequence and cause of cardiomyopathy.

The Ras-like Rab GTPases regulate vesicle transport in endocytosis and exocytosis. We found that cardiac Rabs1, 4, and 6 are upregulated in a dilated cardiomyopathy model overexpressing beta(2)-adrenergic receptors. To determine if increased Rab GTPase expression can contribute to cardiomyopathy, we transgenically overexpressed in mouse hearts prototypical Rab1a, the small G protein that regulates vesicle transport from endoplasmic reticulum to and through Golgi. In multiple independent mouse lines, Rab1a overexpression caused cardiac hypertrophy that progressed in a time- and transgene dose-dependent manner to heart failure. Isolated cardiac myocytes were hypertrophied and exhibited contractile depression with impaired calcium reuptake. Ultrastructural analysis revealed enlarged Golgi stacks and increased transitional vesicles in ventricular myocytes, with increased secretory atrial natriuretic peptide granules and degenerative myelin figures in atrial myocytes; immunogold studies localized Rab1a to these abnormal vesicular structures. A survey of hypertrophy signaling molecules revealed increased protein kinase C (PKC) alpha and delta, and confocal microscopy showed abnormal subcellular distribution of PKCalpha in Rab1a transgenics. These results indicate that increased expression of Rab1 GTPase in myocardium distorts subcellular localization of proteins and is sufficient to cause cardiac hypertrophy and failure.

Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC.

Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported. Here, deltaPKC-selective activator and inhibitor peptides were designed rationally, based on molecular modeling and structural homology analyses. Together with previously identified activator and inhibitor peptides of epsilonPKC, deltaPKC peptides were used to identify cardiac functions of these isozymes. In isolated cardiomyocytes, perfused hearts, and transgenic mice, deltaPKC and epsilonPKC had opposing actions on protection from ischemia-induced damage. Specifically, activation of epsilonPKC caused cardioprotection whereas activation of deltaPKC increased damage induced by ischemia in vitro and in vivo. In contrast, deltaPKC and epsilonPKC caused identical nonpathological cardiac hypertrophy; activation of either isozyme caused nonpathological hypertrophy of the heart. These results demonstrate that two related PKC isozymes have both parallel and opposing effects in the heart, indicating the danger in the use of therapeutics with nonselective isozyme inhibitors and activators. Moreover, reduction in cardiac damage caused by ischemia by perfusion of selective regulator peptides of PKC through the coronary arteries constitutes a major step toward developing a therapeutic agent for acute cardiac ischemia.

Divergent transcriptional responses to independent genetic causes of cardiac hypertrophy.

To define molecular mechanisms of cardiac hypertrophy, genes whose expression was perturbed by any of four different transgenic mouse hypertrophy models [protein kinase C-epsilon activation peptide (PsiepsilonRACK), calsequestrin (CSQ), calcineurin (CN), and Galpha(q)] were compared by DNA microarray analyses using the approximately 8,800 genes present on the Incyte mouse GEM1. The total numbers of regulated genes (tens to hundreds) correlated with phenotypic severity of the model (Galpha(q) > CN > CSQ > PsiepsilonRACK), but demonstrated that no single gene was consistently upregulated. Of the three models exhibiting pathological hypertrophy, only atrial natriuretic peptide was consistently upregulated, suggesting that transcriptional alterations are highly specific to individual genetic causes of hypertrophy. However, hierarchical-tree and K-means clustering analyses revealed that subsets of the upregulated genes did exhibit coordinate regulatory patterns that were unique or overlapping across the different hypertrophy models. One striking set consisted of apoptotic genes uniquely regulated in the apoptosis-prone Galpha(q) model. Thus, rather than identifying a single common hypertrophic cardiomyopathy gene program, these data suggest that extensive groups of genes may be useful for the prediction of specific underlying genetic determinants and condition-specific therapeutic approaches.

Mouse model of desmin-related cardiomyopathy.

BACKGROUND: The consequence of upregulation of desmin in the heart is unknown. Mutations in desmin have been linked to desmin-related myopathy (DRM), which is characterized by abnormal intrasarcoplasmic accumulation of desmin, but direct causative evidence that a desmin mutation leads to aberrant intrasarcoplasmic desmin accumulation, aggregation, and cardiomyopathy is lacking. METHODS AND RESULTS: Multiple transgenic mouse lines that expressed either murine wild-type desmin or a 7-amino acid deletion (R173 through E179) desmin (D7-des) mutation linked to DRM were made. The distribution of desmin protein was unchanged, and no overt phenotype was detected in the wild-type desmin transgenic mice. In contrast, the D7-des mouse heart showed aberrant intrasarcoplasmic and electron-dense granular filamentous aggregates that were desmin-positive and characteristic of human DRM. The desmin filament network was significantly disrupted, and myofibril alignment was visibly compromised. Although systolic function at the whole-organ level was substantially conserved in the young adult animals, the ability of the heart to respond to beta-agonist stimulation, as measured in the intact animal, was significantly blunted. CONCLUSIONS: Upregulation of desmin protein at moderate levels is not detrimental. However, the D7-des mutation is dominant negative, and expression of the mutant protein leads to the appearance of aggregates that are characteristic of and diagnostic for human desmin-related cardiomyopathy.

Superinhibition of sarcoplasmic reticulum function by phospholamban induces cardiac contractile failure.

To determine whether selective impairment of cardiac sarcoplasmic reticulum (SR) Ca(2+) transport may drive the progressive functional deterioration leading to heart failure, transgenic mice, overexpressing a phospholamban Val(49) --> Gly mutant (2-fold), which is a superinhibitor of SR Ca(2+)-ATPase affinity for Ca(2+), were generated, and their cardiac phenotype was examined longitudinally. At 3 months of age, the increased EC(50) level of SR Ca(2+) uptake for Ca(2+) (0.67 +/- 0.09 microm) resulted in significantly higher depression of cardiomyocyte rates of shortening (57%), relengthening (31%), and prolongation of the Ca(2+) signal decay time (165%) than overexpression (2-fold) of wild type phospholamban (68%, 64%, and 125%, respectively), compared with controls (100%). Echocardiography also revealed significantly depressed function and impaired beta-adrenergic responses in mutant hearts. The depressed contractile parameters were associated with left ventricular remodeling, recapitulation of fetal gene expression, and hypertrophy, which progressed to dilated cardiomyopathy with interstitial tissue fibrosis and death by 6 months in males. Females also had ventricular hypertrophy at 3 months but exhibited normal systolic function up to 12 months of age. These results suggest a causal relationship between defective SR Ca(2+) cycling and cardiac remodeling leading to heart failure, with a gender-dependent influence on the time course of these alterations.

Cytoplasmic signaling pathways that regulate cardiac hypertrophy.

This review discusses the rapidly progressing field of cardiomyocyte signal transduction and the regulation of the hypertrophic response. When stimulated by a wide array of neurohumoral factors or when faced with an increase in ventricular-wall tension, individual cardiomyocytes undergo hypertrophic growth as an adaptive response. However, sustained cardiac hypertrophy is a leading predictor of future heart failure. A growing number of intracellular signaling pathways have been characterized as important transducers of the hypertrophic response, including specific G protein isoforms, low-molecular-weight GTPases (Ras, RhoA, and Rac), mitogen-activated protein kinase cascades, protein kinase C, calcineurin, gp130-signal transducer and activator of transcription, insulin-like growth factor I receptor pathway, fibroblast growth factor and transforming growth factor beta receptor pathways, and many others. Each of these signaling pathways has been implicated as a hypertrophic transducer, which collectively suggests an emerging paradigm whereby multiple pathways operate in concert to orchestrate a hypertrophic response

Interactions between phospholamban and beta-adrenergic drive may lead to cardiomyopathy and early mortality.

BACKGROUND: Relieving the inhibition of sarcoplasmic reticular function by phospholamban is a major target of beta-adrenergic stimulation. Chronic beta-adrenergic receptor activity has been suggested to be detrimental, on the basis of transgenic overexpression of the receptor or its signaling effectors. However, it is not known whether physiological levels of sympathetic tone, in the absence of preexisting heart failure, are similarly detrimental. METHODS AND RESULTS: Transgenic mice overexpressing phospholamban at 4-fold normal levels were generated, and at 3 months, they exhibited mildly depressed ventricular contractility without heart failure. As expected, transgenic cardiomyocyte mechanics and calcium kinetics were depressed, but isoproterenol reversed the inhibitory effects of phospholamban on these parameters. In vivo cardiac function was substantially depressed by propranolol administration, suggesting enhanced sympathetic tone. Indeed, plasma norepinephrine levels and the phosphorylation status of phospholamban were elevated, reflecting increased adrenergic drive in transgenic hearts. On aging, the chronic enhancement of adrenergic tone was associated with a desensitization of adenylyl cyclase (which intensified the inhibitory effects of phospholamban), the development of overt heart failure, and a premature mortality. CONCLUSIONS: The unique interaction between phospholamban and increased adrenergic drive, elucidated herein, provides the first evidence that compensatory increases in catecholamine stimulation can, even in the absence of preexisting heart failure, be a primary causative factor in the development of cardiomyopathy and early mortality.

RGS4 reduces contractile dysfunction and hypertrophic gene induction in Galpha q overexpressing mice.

The intrinsic GTPase activity of Galpha q is low, and RGS proteins which activate GTPase are expressed in the heart; however, their functional relevance in vivo is unknown. Transgenic mice with cardiac-specific overexpression of Galpha q in myocardium exhibit cardiac hypertrophy, enhanced PKC xi membrane translocation, embryonic gene expression, and depressed cardiac contractility. We recently reported that transgenic mice with cardiac-specific expression of RGS4, a Galpha q and Galpha i GTPase activator, exhibit decreased left ventricular hypertrophy and ANF induction in response to pressure overload. To test the hypothesis that RGS4 can act as a Galpha q-specific GTPase activating protein (GAP) in the in vivo heart, dual transgenic Galpha q-40xRGS4 mice were generated to determine if RGS4 co-expression would ameliorate the Galpha q-40 phenotype. At age 4 weeks, percent fractional shortening was normalized in dual transgenic mice as was left ventricular internal dimension and posterior and septal wall thicknesses. PKC xi membrane translocation and ANF and alpha -skeletal actin mRNA levels were also normalized. Compound transgenic mice eventually developed depressed cardiac contractility that was evident by 9 weeks of age. These studies establish for the first time a role for RGS4 as a GAP for Galpha q in the in vivo heart, and demonstrate that its regulated expression can have pathophysiologic consequences.

Rescue of contractile parameters and myocyte hypertrophy in calsequestrin overexpressing myocardium by phospholamban ablation.

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca(2+)-induced Ca(2+) release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca(2+) storage, assessed by caffeine-induced Na(+)-Ca(2+) exchanger currents. The inactivation time of the L-type Ca(2+) current (I(Ca)), which has an inverse correlation with Ca(2+)-induced SR Ca(2+) release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability of I(Ca) to trigger SR Ca(2+) release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and alpha-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.

Epsilon protein kinase C in pathological myocardial hypertrophy. Analysis by combined transgenic expression of translocation modifiers and Galphaq.

The epsilon isoform of protein kinase C (PKC) has a critical cardiotrophic function in normal postnatal developing heart as demonstrated by cardiac-specific transgenic expression of epsilonPKC-selective translocation inhibitor (epsilonV1) and activator (psiepsilonRACK) peptides (Mochly-Rosen, D., Wu, G., Hahn, H., Osinska, H., Liron, T., Lorenz, J. N., Robbins, J., and Dorn, G. W., II (2000) Circ. Res. 86, 1173-1179). To define the role of epsilonPKC signaling in pathological myocardial hypertrophy, epsilonV1 or psiepsilonRACK were co-expressed in mouse hearts with Galpha(q), a PKC-linked hypertrophy signal transducer. Compared with Galpha(q) overexpression alone, co-expression of psiepsilonRACK with Galpha(q) increased epsilonPKC particulate partitioning by 30 +/- 2%, whereas co-expression of epsilonV1 with Galpha(q) reduced particulate-associated epsilonPKC by 22 +/- 1%. Facilitation of epsilonPKC translocation by psiepsilonRACK in Galpha(q) mice improved cardiac contractile function measured as left ventricular fractional shortening (30 +/- 3% Galpha(q) versus 43 +/- 2% psiepsilonRACK/Galpha(q), p < 0.05). Conversely, inhibition of epsilonPKC by epsilonV1 modified the Galpha(q) nonfailing hypertrophy phenotype to that of a lethal dilated cardiomyopathy. These opposing effects of epsilonPKC translocation activation and inhibition in Galpha(q) hypertrophy indicate that epsilonPKC signaling is a compensatory event in myocardial hypertrophy, rather than a pathological event, and support the possible therapeutic efficacy of selective epsilonPKC translocation enhancement in cardiac insufficiency.

Cardiotrophic effects of protein kinase C epsilon: analysis by in vivo modulation of PKCepsilon translocation.

Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (epsilonV1) and translocation activator (psiepsilonRACK [receptors for activated C kinase]) specifically targeting PKCepsilon. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCepsilon signaling. As expected for a PKCepsilon RACK binding peptide, confocal microscopy showed that epsilonV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCepsilon by epsilonV1 at lower expression levels upregulated alpha-skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, epsilonV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCepsilon translocation with psiepsilonRACK resulted in selectively increased beta myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCepsilon signaling in normal postnatal maturational myocardial development and suggest the potential for PKCepsilon activators to stimulate "physiological" cardiomyocyte growth.

Polymorphisms of the beta(2)-adrenergic receptor determine exercise capacity in patients with heart failure.

The beta(2)-adrenergic receptor (beta(2)AR) exists in multiple polymorphic forms with different characteristics. Their relevance to heart failure (HF) physiology is unknown. Cardiopulmonary exercise testing was performed on 232 compensated HF patients with a defined beta(2)AR genotype. Patients with the uncommon Ile164 polymorphism had a lower peak VO(2) (15.0+/-0.9 mL. kg(-1). min(-1)) than did patients with Thr164 (17.9+/-0.9 mL. kg(-1). min(-1), P<0.0001). The percentage achieved of predicted peak VO(2) was also lower in patients with Ile164 (62. 3+/-4.5% versus 71.5+/-5.1%, P=0.045). The relative risk of a patient having a VO(2)

Early and delayed consequences of beta(2)-adrenergic receptor overexpression in mouse hearts: critical role for expression level.

BACKGROUND: Transgenic cardiac beta(2)-adrenergic receptor (AR) overexpression has resulted in enhanced signaling and cardiac function in mice, whereas relatively low levels of transgenically expressed G(alphas) or beta(1)AR have resulted in phenotypes of ventricular failure. Potential relationships between the levels of betaAR overexpression and biochemical, molecular, and physiological consequences have not been reported. METHODS AND RESULTS: We generated transgenic mice expressing beta(2)AR at 3690, 7120, 9670, and 23 300 fmol/mg in the heart, representing 60, 100, 150, and 350 times background betaAR expression. All lines showed enhanced basal adenylyl cyclase activation but a decrease in forskolin- and NaF-stimulated adenylyl cyclase activities. Mice of the highest-expressing line developed a rapidly progressive fibrotic dilated cardiomyopathy and died of heart failure at 25+/-1 weeks of age. The 60-fold line exhibited enhanced basal cardiac function without increased mortality when followed for 1 year, whereas 100-fold overexpressors developed a fibrotic cardiomyopathy and heart failure, with death occurring at 41+/-1 weeks of age. Adenylyl cyclase activation did not correlate with early or delayed decompensation. Propranolol administration reduced baseline +dP/dt(max) to nontransgenic levels in all beta(2)AR transgenics except the 350-fold overexpressors, indicating that spontaneous activation of beta(2)AR was present at this level of expression. CONCLUSIONS: These data demonstrate that the heart tolerates enhanced contractile function via 60-fold beta(2)AR overexpression without detriment for a period of >/=1 year and that higher levels of expression result in either aggressive or delayed cardiomyopathy. The consequences for enhanced betaAR function in the heart appear to be highly dependent on which signaling elements are increased and to what extent.