Molecular inotropy mediated by cardiac miR-based PDE4D/PRKAR1α/phosphoprotein signaling.

Molecular inotropy refers to cardiac contractility that can be modified to affect overall heart pump performance. Here we show evidence of a new molecular pathway for positive inotropy by a cardiac-restricted microRNA (miR). We report enhanced cardiac myocyte performance by acute titration of cardiac myosin-embedded miR-208a. The observed positive effect was independent of host gene myosin effects with evidence of negative regulation of cAMP-specific 3',5'-cyclic phosphodiesterase 4D (PDE4D) and the regulatory subunit of PKA (PRKAR1α) content culminating in PKA-site dependent phosphorylation of cardiac troponin I (cTnI) and phospholamban (PLN). Further, acute inhibition of miR-208a in adult myocytes in vitro increased PDE4D expression causing reduced isoproterenol-mediated phosphorylation of cTnI and PLN. Next, rAAV-mediated miR-208a gene delivery enhanced heart contractility and relaxation parameters in vivo. Finally, acute inducible increases in cardiac miR-208a in vivo reduced PDE4D and PRKAR1α, with evidence of increased content of several complementary miRs harboring the PDE4D recognition sequence. Physiologically, this resulted in significant cardiac cTnI and PLN phosphorylation and improved heart performance in vivo. As phosphorylation of cTnI and PLN is critical to myocyte function, titration of miR-208a represents a potential new mechanism to enhance myocardial performance via the PDE4D/PRKAR1α/PKA phosphoprotein signaling pathway.

Uncoupling of increased cellular oxidative stress and myocardial ischemia reperfusion injury by directed sarcolemma stabilization.

Myocardial ischemia/reperfusion (I/R) injury is a major clinical problem leading to cardiac dysfunction and myocyte death. It is widely held that I/R causes damage to membrane phospholipids, and is a significant mechanism of cardiac I/R injury. Molecular dissection of sarcolemmal damage in I/R, however, has been difficult to address experimentally. We studied here cardiac I/R injury under conditions targeting gain- or loss-of sarcolemma integrity. To implement gain-in-sarcolemma integrity during I/R, synthetic copolymer-based sarcolemmal stabilizers (CSS), including Poloxamer 188 (P188), were used as a tool to directly stabilize the sarcolemma. Consistent with the hypothesis of sarcolemmal stabilization, cellular markers of necrosis and apoptosis evident in untreated myocytes were fully blocked in sarcolemma stabilized myocytes. Unexpectedly, sarcolemmal stabilization of adult cardiac myocytes did not affect the status of myocyte-generated oxidants or lipid peroxidation in two independent assays. We also investigated the loss of sarcolemmal integrity using two independent genetic mouse models, dystrophin-deficient mdx or dysferlin knockout (Dysf KO) mice. Both models of sarcolemmal loss-of-function were severely affected by I/R injury ex vivo, and this was lessened by CSS. In vivo studies also showed that infarct size was significantly reduced in CSS-treated hearts. Mechanistically, these findings support a model whereby I/R-mediated increased myocyte oxidative stress is uncoupled from myocyte injury. Because the sarcolemma stabilizers used here do not transit across the myocyte membrane this is evidence that intracellular targets of oxidants are not sufficiently altered to affect cell death when sarcolemma integrity is preserved by synthetic stabilizers. These findings, in turn, suggest that sarcolemma destabilization, and consequent Ca(2+) mishandling, as a focal initiating mechanism underlying myocardial I/R injury.

Direct, differential effects of tamoxifen, 4-hydroxytamoxifen, and raloxifene on cardiac myocyte contractility and calcium handling.

Tamoxifen (Tam), a selective estrogen receptor modulator, is in wide clinical use for the treatment and prevention of breast cancer. High Tam doses have been used for treatment of gliomas and cancers with multiple drug resistance, but long QT Syndrome is a side effect. Tam is also used experimentally in mice for inducible gene knockout in numerous tissues, including heart; however, the potential direct effects of Tam on cardiac myocyte mechanical function are not known. The goal of this study was to determine the direct, acute effects of Tam, its active metabolite 4-hydroxytamoxifen (4OHT), and related drug raloxifene (Ral) on isolated rat cardiac myocyte mechanical function and calcium handling. Tam decreased contraction amplitude, slowed relaxation, and decreased Ca²âº transient amplitude. Effects were primarily observed at 5 and 10 µM Tam, which is relevant for high dose Tam treatment in cancer patients as well as Tam-mediated gene excision in mice. Myocytes treated with 4OHT responded similarly to Tam-treated cells with regard to both contractility and calcium handling, suggesting an estrogen-receptor independent mechanism is responsible for the effects. In contrast, Ral increased contraction and Ca²âº transient amplitudes. At 10 µM, all drugs had a time-dependent effect to abolish cellular contraction. In conclusion, Tam, 4OHT, and Ral adversely and differentially alter cardiac myocyte contractility and Ca²âº handling. These findings have important implications for understanding the Tam-induced cardiomyopathy in gene excision studies and may be important for understanding effects on cardiac performance in patients undergoing high-dose Tam therapy.

Calcium mishandling in diastolic dysfunction: mechanisms and potential therapies.

Diastolic dysfunction is characterized by slow or incomplete relaxation of the ventricles during diastole, and is an important contributor to heart failure pathophysiology. Clinical symptoms include fatigue, shortness of breath, and pulmonary and peripheral edema, all contributing to decreased quality of life and poor prognosis. There are currently no therapies available that directly target the heart pump defects in diastolic function. Calcium mishandling is a hallmark of heart disease and has been the subject of a large body of research. Efforts are ongoing in a number of gene therapy approaches to normalize the function of calcium handling proteins such as sarcoplasmic reticulum calcium ATPase. An alternative approach to address calcium mishandling in diastolic dysfunction is to introduce calcium buffers to facilitate relaxation of the heart. Parvalbumin is a calcium binding protein found in fast-twitch skeletal muscle and not normally expressed in the heart. Gene transfer of parvalbumin into normal and diseased cardiac myocytes increases relaxation rate but also markedly decreases contraction amplitude. Although parvalbumin binds calcium in a delayed manner, it is not delayed enough to preserve full contractility. Factors contributing to the temporal nature of calcium buffering by parvalbumin are discussed in relation to remediation of diastolic dysfunction. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Mesencephalic astrocyte-derived neurotrophic factor is an ischemia-inducible secreted endoplasmic reticulum stress response protein in the heart.

The endoplasmic reticulum (ER) stress response (ERSR) is activated when folding of nascent proteins in the ER lumen is impeded. Myocardial ischemia was recently shown to activate the ERSR; however, the role of this complex signaling system in the heart is not well understood. ER stress activates the transcription factor ATF6, which induces expression of proteins targeted to the ER, where they restore protein folding, thus fostering cytoprotection. We previously developed a transgenic mouse line that expresses a conditionally activated form of ATF6 in the heart. In this mouse line, ATF6 activation decreased ischemic damage in an ex vivo model of myocardial ischemia/reperfusion and induced numerous genes, including mesencephalic astrocyte-derived neurotrophic factor (MANF). In the present study, MANF expression was shown to be induced in cardiac myocytes and in other cell types in the hearts of mice subjected to in vivo myocardial infarction. Additionally, simulated ischemia induced MANF in an ATF6-dependent manner in neonatal rat ventricular myocyte cultures. In contrast to many other ER-resident ERSR proteins, MANF lacks a canonical ER-retention sequence, consistent with our finding that MANF was readily secreted from cultured cardiac myocytes. Knockdown of endogenous MANF with micro-RNA increased cell death upon simulated ischemia/reperfusion, whereas addition of recombinant MANF to media protected cultured cardiac myocytes from simulated ischemia/reperfusion-mediated death. Thus, a possible function of the ERSR in the heart is the ischemia-mediated induction of secreted proteins, such as MANF, that can function in an autocrine/paracrine manner to modulate myocardial damage from ER stresses, including ischemia.

Coordination of growth and endoplasmic reticulum stress signaling by regulator of calcineurin 1 (RCAN1), a novel ATF6-inducible gene.

Exposing cells to conditions that modulate growth can impair endoplasmic reticulum (ER) protein folding, leading to ER stress and activation of the transcription factor, ATF6. ATF6 binds to ER stress response elements in target genes, inducing expression of proteins that enhance the ER protein folding capacity, which helps overcome the stress and foster survival. To examine the mechanism of ATF6-mediated survival in vivo, we developed a transgenic mouse model that expresses a novel conditionally activated form of ATF6. We previously showed that activating ATF6 protected the hearts of ATF6 transgenic mice from ER stresses. In the present study, transcript profiling identified modulatory calcineurin interacting protein-1 (MCIP1), also known as regulator of calcineurin 1 (RCAN1), as a novel ATF6-inducible gene that encodes a known regulator of calcineurin/nuclear factor of activated T cells (NFAT)-mediated growth and development in many tissues. The ability of ATF6 to induce RCAN1 in vivo was replicated in cultured cardiac myocytes, where adenoviral (AdV)-mediated overexpression of activated ATF6 induced the RCAN1 promoter, up-regulated RCAN1 mRNA, inhibited calcineurin phosphatase activity, and exerted a striking growth modulating effect that was inhibited by RCAN1-targeted small interfering RNA. These results demonstrate that RCAN1 is a novel ATF6 target gene that may coordinate growth and ER stress signaling pathways. By modulating growth, RCAN1 may reduce the need for ER protein folding, thus helping to overcome the stress and enhance survival. Moreover, these results suggest that RCAN1 may also be a novel integrator of growth and ER stress signaling in many other tissues that depend on calcineurin/NFAT signaling for optimal growth and development.

Pim-1 regulates cardiomyocyte survival downstream of Akt.

The serine-threonine kinases Pim-1 and Akt regulate cellular proliferation and survival. Although Akt is known to be a crucial signaling protein in the myocardium, the role of Pim-1 has been overlooked. Pim-1 expression in the myocardium of mice decreased during postnatal development, re-emerged after acute pathological injury in mice and was increased in failing hearts of both mice and humans. Cardioprotective stimuli associated with Akt activation induced Pim-1 expression, but compensatory increases in Akt abundance and phosphorylation after pathological injury by infarction or pressure overload did not protect the myocardium in Pim-1-deficient mice. Transgenic expression of Pim-1 in the myocardium protected mice from infarction injury, and Pim-1 expression inhibited cardiomyocyte apoptosis with concomitant increases in Bcl-2 and Bcl-X(L) protein levels, as well as in Bad phosphorylation levels. Relative to nontransgenic controls, calcium dynamics were significantly enhanced in Pim-1-overexpressing transgenic hearts, associated with increased expression of SERCA2a, and were depressed in Pim-1-deficient hearts. Collectively, these data suggest that Pim-1 is a crucial facet of cardioprotection downstream of Akt.

Alterations in oxidative phosphorylation complex proteins in the hearts of transgenic mice that overexpress the p38 MAP kinase activator, MAP kinase kinase 6.

Ischemia-reperfusion (I/R) has critical consequences in the heart. Recent studies on the functions of I/R-activated kinases, such as p38 mitogen-activated protein kinase (MAPK), showed that I/R injury is reduced in the hearts of transgenic mice that overexpress the p38 MAPK activator MAPK kinase 6 (MKK6). This protection may be fostered by changes in the levels of many proteins not currently known to be regulated by p38. To examine this possibility, we employed the multidimensional protein identification technology MudPIT to characterize changes in levels of proteins in MKK6 transgenic mouse hearts, focusing on proteins in mitochondria, which play key roles in mediating I/R injury in the heart. Of the 386 mitochondrial proteins identified, the levels of 58 were decreased, while only 2 were increased in the MKK6 transgenic mouse hearts. Among those that were decreased were 21 mitochondrial oxidative phosphorylation complex proteins, which was unexpected because p38 is not known to mediate such decreases. Immunoblotting verified that proteins in each of the five oxidative phosphorylation complexes were reduced in MKK6 mouse hearts. On assessing functional consequences of these reductions, we found that MKK6 mouse heart mitochondria exhibited 50% lower oxidative respiration and I/R-mediated reactive oxygen species (ROS) generation, both of which are predicted consequences of decreased oxidative phosphorylation complex proteins. Thus the cardioprotection observed in MKK6 transgenic mouse hearts may be partly due to decreased electron transport, which is potentially beneficial, because damaging ROS are known to be generated by mitochondrial complexes I and III during reoxygenation.

Endoplasmic reticulum stress gene induction and protection from ischemia/reperfusion injury in the hearts of transgenic mice with a tamoxifen-regulated form of ATF6.

Ischemia/reperfusion (I/R) affects the integrity of the endoplasmic reticulum (ER), the site of synthesis and folding of numerous proteins. Therefore, I/R may activate the unfolded protein response (UPR), resulting in the induction of a collection of ER stress proteins, many of which are protective and function to resolve the ER stress. In this study, we showed that when mouse hearts were subjected to ex vivo I/R, the levels of 2 ER stress-inducible markers of the UPR, the ER-targeted cytoprotective chaperones glucose-regulated proteins 78 and 94 (GRP78 and GRP94), were increased, consistent with I/R-mediated UPR activation in the heart. The UPR-mediated activation of ATF6 (Activation of Transcription Factor 6) induces cytoprotective ER stress proteins, including GRP78 and GRP94. To examine whether ATF6 protects the myocardium from I/R injury in the heart, we generated transgenic (TG) mice featuring cardiac-restricted expression of a novel tamoxifen-activated form of ATF6, ATF6-MER. When NTG and ATF6-MER TG mice were treated with or without tamoxifen for 5 days, only the hearts from the tamoxifen-treated TG mice exhibited increased levels of many ER stress-inducible mRNAs and proteins; for example, GRP78 and GRP94 transcript levels were increased by 8- and 15-fold, respectively. The tamoxifen-treated TG mouse hearts also exhibited better functional recovery from ex vivo I/R, as well as significantly reduced necrosis and apoptosis. These results suggest that the UPR is activated in the heart during I/R and that, as a result, the ATF6 branch of the UPR may induce expression of proteins that can function to reduce I/R injury.

Overexpression of mitogen-activated protein kinase kinase 6 in the heart improves functional recovery from ischemia in vitro and protects against myocardial infarction in vivo.

The mitogen-activated protein kinases (MAPK) have been the subject of many studies to identify signaling pathways that promote cell survival or death. In cultured cardiac myocytes, p38 MAPK promotes cell survival or death depending on whether it is activated by mitogen-activated protein kinase kinase 6 (MKK6) or MKK3, respectively. The objectives of the current study were to examine the effects of MKK6-mediated p38 activation in the heart in vivo. Accordingly, we generated transgenic (TG) mice that overexpress wild type MKK6 in a cardiac-restricted manner. Although p38 was about 17-fold more active in TG than non-transgenic (NTG) mouse hearts, TG mouse hearts were morphologically and functionally similar to those of NTG littermates. However, upon transient ischemia followed by reperfusion, the MKK6 TG mouse hearts exhibited significantly better functional recovery and less injury than NTG mouse hearts. Because MKK6 increases levels of the protective small heat shock protein, alpha B-crystallin (alpha BC), in cultured cardiac myocytes, we examined alpha BC levels in the mouse hearts. The level of alpha BC was 2-fold higher in MKK6 TG than NTG mouse hearts. Moreover, ischemia followed by reperfusion induced a 6.4-fold increase in alpha BC levels in the mitochondrial fractions of TG mouse hearts but no increase in alpha BC levels in any of the other fractions analyzed. These alterations in alpha BC expression and localization suggest possible mechanisms of cardioprotection in MKK6 TG mouse hearts.