John P. Morrow

Temsirolimus Activates Autophagy and Ameliorates Cardiomyopathy Caused by Lamin A/C Gene Mutation

Cardiomyopathy arising from lamin A/C mutations in mice can be corrected by enhancing autophagy through blockade of mTOR with temsirolimus. Taking a Bite Out of Cardiomyopathy Mutations that affect nuclear lamins, which form the mesh-like layer inside the nuclear membrane, cause a surprising variety of disorders, together termed laminopathies. Physically stressed tissues such as muscle, where maintenance of nuclear envelope stability is important, are often affected—indeed, lamins are found exclusively in animals, presumably as an adaptation to mobility. Although exactly how altered lamins cause disease is not yet clear, cell signaling pathways seem to be affected. The new work by Choi et al. sheds light on a major signaling pathway affected in one laminopathy and indicates possible therapeutic agents. The most common laminopathy, dilated cardiomyopathy, is caused by certain mutations in the A-type lamin gene LMNA. Here, the heart is enlarged and weakened, often leading to heart failure and premature death. Because altered signaling by the kinase AKT has been associated with other forms of cardiomyopathy, Choi and colleagues examined such signaling in a mouse model in which cardiomyopathy is caused by an Lmna point mutation. They found abnormally enhanced AKT activation in the hearts of these mice, even before the onset of detectable cardiomyopathy, as well as hyperactivation of an AKT target, the kinase mammalian target of rapamycin (mTOR). Treatment with temsirolimus, a derivative of the mTOR complex 1 (mTORC1) inhibitor rapamycin, reduced mTOR activation and prevented cardiac defects. mTORC1 inhibits autophagy, a highly regulated process in which cellular components are degraded to maintain tissue homeostasis. The authors found that autophagy was deficient in the hearts of the diseased mice and that temsirolimus treatment reactivated autophagy. The enhanced autophagy correlated with improved heart function. Choi and co-workers also observed enhanced AKT activation in heart tissue from patients with dilated cardiomyopathy caused by mutations in LMNA, supporting the idea that mTORC1 inhibitors might be useful for treating this condition in humans. This concept is reinforced by a related work described by Ramos et al. in this issue. Mutations in the lamin A/C gene (LMNA), which encodes A-type lamins, cause a diverse range of diseases collectively called laminopathies, the most common of which is dilated cardiomyopathy. Emerging evidence suggests that LMNA mutations cause disease by altering cell signaling pathways, but the specific mechanisms are poorly understood. We show that the AKT–mammalian target of rapamycin pathway is hyperactivated in hearts of mice with cardiomyopathy caused by Lmna mutation and that in vivo administration of the rapamycin analog temsirolimus prevents deterioration of cardiac function. We also show defective autophagy in hearts of these mice and demonstrate that improvement in heart function induced by pharmacological interventions is correlated with enhanced autophagy. These findings provide a rationale for treatment of LMNA cardiomyopathy with rapalogs and implicate defective autophagy as a pathogenic mechanism of cardiomyopathy arising from LMNA mutation.

Enhanced Efferocytosis of Apoptotic Cardiomyocytes Through Myeloid-Epithelial-Reproductive Tyrosine Kinase Links Acute Inflammation Resolution to Cardiac Repair After Infarction

Rationale: Efficient clearance of apoptotic cells (efferocytosis) is a prerequisite for inflammation resolution and tissue repair. After myocardial infarction, phagocytes are recruited to the heart and promote clearance of dying cardiomyocytes. The molecular mechanisms of efferocytosis of cardiomyocytes and in the myocardium are unknown. The injured heart provides a unique model to examine relationships between efferocytosis and subsequent inflammation resolution, tissue remodeling, and organ function. Objective: We set out to identify mechanisms of dying cardiomyocyte engulfment by phagocytes and, for the first time, to assess the causal significance of disrupting efferocytosis during myocardial infarction. Methods and Results: In contrast to other apoptotic cell receptors, macrophage myeloid-epithelial-reproductive tyrosine kinase was necessary and sufficient for efferocytosis of cardiomyocytes ex vivo. In mice, Mertk was specifically induced in Ly6cLO myocardial phagocytes after experimental coronary occlusion. Mertk deficiency led to an accumulation of apoptotic cardiomyocytes, independently of changes in noncardiomyocytes, and a reduced index of in vivo efferocytosis. Importantly, suppressed efferocytosis preceded increases in myocardial infarct size and led to delayed inflammation resolution and reduced systolic performance. Reduced cardiac function was reproduced in chimeric mice deficient in bone marrow Mertk; reciprocal transplantation of Mertk+/+ marrow into Mertk−/− mice corrected systolic dysfunction. Interestingly, an inactivated form of myeloid-epithelial-reproductive tyrosine kinase, known as solMER, was identified in infarcted myocardium, implicating a natural mechanism of myeloid-epithelial-reproductive tyrosine kinase inactivation after myocardial infarction. Conclusions: These data collectively and directly link efferocytosis to wound healing in the heart and identify Mertk as a significant link between acute inflammation resolution and organ function.

Ventricular arrhythmias and implantable cardioverter-defibrillator therapy in patients with continuous-flow left ventricular assist devices: Need for primary prevention?

OBJECTIVES This study sought to evaluate the prevalence and significance of ventricular arrhythmia (VA) and the role of an implantable cardioverter-defibrillator (ICD) in patients supported by a continuous-flow left ventricular assist device (CF-LVAD). BACKGROUND VAs are common in patients supported by CF-LVADs but prospective data to support the routine use of ICDs in these patients are lacking. METHODS All patients supported by long-term CF-LVAD receiving care at our institution were enrolled. The ICDs were interrogated at baseline and throughout prospective follow-up. The VA was defined as ventricular tachycardia/fibrillation lasting >30 s or effectively terminated by appropriate ICD tachytherapy. The primary outcome was the occurrence of VA >30 days after CF-LVAD implantation. RESULTS Ninety-four patients were enrolled; 77 had an ICD and 17 did not. Five patients with an ICD had it deactivated or a depleted battery not replaced during the study. Twenty-two patients had a VA >30 days after LVAD implantation. Pre-operative VA was the major predictor of post-operative arrhythmia. Absence of pre-operative VA conferred a low risk of post-operative VA (4.0% vs. 45.5%; p < 0.001). No patients discharged from the hospital without an ICD after CF-LVAD implantation died during 276.2 months of follow-up (mean time without ICD, 12.7 ± 12.3 months). CONCLUSIONS Patients with pre-operative VA are at risk of recurrent VA while on CF-LVAD support and should have active ICD therapy to minimize sustained VA. Patients without pre-operative VA are at low risk and may not need active ICD therapy.

Enhanced Efferocytosis of Apoptotic Cardiomyocytes Through MER Tyrosine Kinase Links Acute Inflammation Resolution to Cardiac Repair After Infarction

Rationale: Efficient clearance of apoptotic cells (efferocytosis) is a prerequisite for inflammation resolution and tissue repair. After myocardial infarction, phagocytes are recruited to the heart and promote clearance of dying cardiomyocytes. The molecular mechanisms of efferocytosis of cardiomyocytes and in the myocardium are unknown. The injured heart provides a unique model to examine relationships between efferocytosis and subsequent inflammation resolution, tissue remodeling, and organ function. Objective: We set out to identify mechanisms of dying cardiomyocyte engulfment by phagocytes and, for the first time, to assess the causal significance of disrupting efferocytosis during myocardial infarction. Methods and Results: In contrast to other apoptotic cell receptors, macrophage myeloid-epithelial-reproductive tyrosine kinase was necessary and sufficient for efferocytosis of cardiomyocytes ex vivo. In mice, Mertk was specifically induced in Ly6cLO myocardial phagocytes after experimental coronary occlusion. Mertk deficiency led to an accumulation of apoptotic cardiomyocytes, independently of changes in noncardiomyocytes, and a reduced index of in vivo efferocytosis. Importantly, suppressed efferocytosis preceded increases in myocardial infarct size and led to delayed inflammation resolution and reduced systolic performance. Reduced cardiac function was reproduced in chimeric mice deficient in bone marrow Mertk; reciprocal transplantation of Mertk+/+ marrow into Mertk−/− mice corrected systolic dysfunction. Interestingly, an inactivated form of myeloid-epithelial-reproductive tyrosine kinase, known as solMER, was identified in infarcted myocardium, implicating a natural mechanism of myeloid-epithelial-reproductive tyrosine kinase inactivation after myocardial infarction. Conclusions: These data collectively and directly link efferocytosis to wound healing in the heart and identify Mertk as a significant link between acute inflammation resolution and organ function.

Protein kinase C isoforms differentially phosphorylate Ca(v)1.2 alpha(1c).

The regulation of Ca(2+) influx through the phosphorylation of the L-type Ca(2+) channel, Ca(v)1.2, is important for the modulation of excitation-contraction (E-C) coupling in the heart. Ca(v)1.2 is thought to be the target of multiple kinases that mediate the signals of both the renin-angiotensin and sympathetic nervous systems. Detailed biochemical information regarding the protein phosphorylation reactions involved in the regulation of Ca(v)1.2 is limited. The protein kinase C (PKC) family of kinases can modulate cardiac contractility in a complex manner, such that contractility is either enhanced or depressed and relaxation is either accelerated or slowed. We have previously reported that Ser(1928) in the C-terminus of alpha(1c) was a target for PKCalpha, -zeta, and -epsilon phosphorylation. Here, we report the identification of seven PKC phosphorylation sites within the alpha(1c) subunit. Using phospho-epitope specific antibodies to Ser(1674) and Ser(1928), we demonstrate that both sites within the C-terminus are phosphorylated in HEK cells in response to PMA. Phosphorylation was inhibited with a PKC inhibitor, bisindolylmaleimide. In Langendorff-perfused rat hearts, both Ser(1674) and Ser(1928) were phosphorylated in response to PMA. Phosphorylation of Ser(1674), but not Ser(1928), is PKC isoform specific, as only PKCalpha, -betaI, -betaII, -gamma, -delta, and -theta, but not PKCepsilon, -zeta, and -eta, were able to phosphorylate this site. Our results identify a molecular mechanism by which PKC isoforms can have different effects on channel activity by phosphorylating different residues.

Cardiac L-type calcium channel (Cav1.2) associates with γ subunits

The cardiac voltage‐gated Ca2+ channel, Cav1.2, mediates excitation‐contraction coupling in the heart. The molecular composition of the channel includes the pore‐forming α1 subunit and auxiliary α2/δ‐1 and β subunits. Ca2+ channel γ subunits, of which there are 8 isoforms, consist of 4 transmembrane domains with intracellular N‐ and C‐terminal ends. The γ1 subunit was initially detected in the skeletal muscle Cav1.1 channel complex, modulating current amplitude and activation and inactivation properties. The γ1 subunit also shifts the steady‐state inactivation to more negative membrane potentials, accelerates current inactivation, and increases peak currents, when coexpressed with the cardiac α1c subunit in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. The γ1 subunit is not expressed, however, in cardiac muscle. We sought to determine whether γ subunits that are expressed in cardiac tissue physically associate with and modulate Cav1.2 function. We now demonstrate that γ4, γ6, γ7, and γ8 subunits physically interact with the Cav1.2 complex. The γ subunits differentially modulate Ca2+ channel function when coexpressed with the β1b and α2/δ‐1 subunits in HEK cells, altering both activation and inactivation properties. The effects of γ on Cav1.2 function are dependent on the subtype of β subunit. Our results identify new members of the cardiac Cav1.2 macro‐molecular complex and identify a mechanism by which to increase the functional diversity of Cav1.2 channels.—Yang, L., Katchman, A., Morrow, J. P., Doshi, D., Marx, S. A. Cardiac L‐type calcium channel (CaV1.2) associates with γ subunits. FASEB J. 25, 928–936 (2011). www.fasebj.org

Abnormal p38α mitogen-activated protein kinase signaling in dilated cardiomyopathy caused by lamin A/C gene mutation.

We previously interrogated the transcriptome in heart tissue from Lmna(H222P/H222P) mice, a mouse model of cardiomyopathy caused by lamin A/C gene (LMNA) mutation, and found that the extracellular signal-regulated kinase 1/2 and Jun N-terminal kinase branches of the mitogen-activated protein (MAP) kinase signaling pathway were abnormally hyperactivated prior to the onset of significant cardiac impairment. We have now used an alternative gene expression analysis tool to reanalyze this transcriptome and identify hyperactivation of a third branch of the MAP kinase cascade, p38α signaling. Biochemical analysis of hearts from Lmna(H222P/H222P) mice showed enhanced p38α activation prior to and after the onset of heart disease as well as in hearts from human subjects with cardiomyopathy caused by LMNA mutations. Treatment of Lmna(H222P/H222P) mice with the p38α inhibitor ARRY-371797 prevented left ventricular dilatation and deterioration of fractional shortening compared with placebo-treated mice but did not block the expression of collagen genes involved in cardiac fibrosis. These results demonstrate that three different branches of the MAP kinase signaling pathway with overlapping consequences are involved in the pathogenesis of cardiomyopathy caused by LMNA mutations. They further suggest that pharmacological inhibition of p38α may be useful in the treatment of this disease.

The cytoplasmic domain of the low density lipoprotein (LDL) receptor-related protein, but not that of the LDL receptor, triggers phagocytosis

The macrophage LDL receptor and LDL receptor-related protein (LRP, CD91) mediate the phagocytic-like uptake of atherogenic lipoproteins and apoptotic cells, yet the structural basis of their phagocytic functions is not known. To address this issue, we transfected macrophages with chimeric proteins containing the cytoplasmic tails and transmembrane regions of the LDL receptor or LRP and the ectodomain of CD2, which can bind non-opsonized sheep red blood cells (SRBCs). Macrophages expressing receptors containing the LDL receptor domains were able to bind but not internalize SRBCs. In contrast, macrophages expressing receptors containing the cytoplasmic tail of LRP were able to bind and internalize SRBCs. Chimeras in which the LRP cytoplasmic tail was mutated in two di-leucine motifs and a tyrosine in an NPXYXXL motif were able to endocytose anti-CD2 antibody and bind SRBCs, but SRBC phagocytosis was decreased by 70%. Thus, the phagocytic-like functions of LRP, but not those of the LDL receptor, can be explained by the ability of the LRP cytoplasmic tail to trigger phagocytosis. These findings have important implications for atherogenesis and apoptotic cell clearance and for a fundamental cell biological understanding of how the LDL receptor and LRP function in internalization processes.

Diet-induced obesity causes long QT and reduces transcription of voltage-gated potassium channels

In humans, obesity is associated with long QT, increased frequency of premature ventricular complexes, and sudden cardiac death. The mechanisms of the pro-arrhythmic electrophysiologic remodeling of obesity are poorly understood. We tested the hypothesis that there is decreased expression of voltage-gated potassium channels in the obese heart, leading to long QT. Using implanted telemeters, we found that diet-induced obese (DIO) wild-type mice have impaired cardiac repolarization, demonstrated by long QT, as well as more frequent ventricular ectopy, similar to obese humans. DIO mice have reduced protein and mRNA levels of the potassium channel Kv1.5 caused by a reduction of the transcription factor cyclic AMP response element binding protein (CREB) in DIO hearts. We found that CREB knock-down by siRNA reduces Kv1.5, CREB binds to the Kv1.5 promoter in the heart, and CREB increases transcription of mouse and human Kv1.5 promoters. The reduction in CREB protein during lipotoxicity can be rescued by inhibiting protein kinase D (PKD). Our results identify a mechanism for obesity-induced electrophysiologic remodeling in the heart, namely PKD-induced reduction of CREB, which in turn decreases expression of the potassium channel Kv1.5.

β-Adrenergic Regulation of the L-type Ca2+ Channel does not Require Phosphorylation of α1C Ser1700

Rationale: Sympathetic nervous system triggered activation of protein kinase A, which phosphorylates several targets within cardiomyocytes, augments inotropy, chronotropy, and lusitropy. An important target of &bgr;-adrenergic stimulation is the sarcolemmal L-type Ca2+ channel, CaV1.2, which plays a key role in cardiac excitation–contraction coupling. The molecular mechanisms of &bgr;-adrenergic regulation of CaV1.2 in cardiomyocytes, however, are incompletely known. Recently, it has been postulated that proteolytic cleavage at Ala1800 and protein kinase A phosphorylation of Ser1700 are required for &bgr;-adrenergic modulation of CaV1.2. Objective: To assess the role of Ala1800 in the cleavage of &agr;1C and the role of Ser1700 and Thr1704 in mediating the adrenergic regulation of CaV1.2 in the heart. Methods and Results: Using a transgenic approach that enables selective and inducible expression in mice of FLAG-epitope–tagged, dihydropyridine-resistant CaV1.2 channels harboring mutations at key regulatory sites, we show that adrenergic regulation of CaV1.2 current and fractional shortening of cardiomyocytes do not require phosphorylation of either Ser1700 or Thr1704 of the &agr;1C subunit. The presence of Ala1800 and the 1798NNAN1801 motif in &agr;1C is not required for proteolytic cleavage of the &agr;1C C-terminus, and deletion of these residues did not perturb adrenergic modulation of CaV1.2 current. Conclusions: These results show that protein kinase A phosphorylation of &agr;1C Ser1700 does not have a major role in the sympathetic stimulation of Ca2+ current and contraction in the adult murine heart. Moreover, this new transgenic approach enables functional and reproducible screening of &agr;1C mutants in freshly isolated adult cardiomyocytes in a reliable, timely, cost-effective manner.