Shinichi Iwata

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.

Effects of Continuous-Flow Versus Pulsatile-Flow Left Ventricular Assist Devices on Myocardial Unloading and Remodeling

Background— Continuous-flow left ventricular assist devices (LVAD) are increasingly used for patients with end-stage heart failure (HF). We analyzed the effects of ventricular decompression by continuous-flow versus pulsatile-flow LVADs on myocardial structure and function in this population. Methods and Results— Sixty-one patients who underwent LVAD implantation as bridge-to-transplant were analyzed (pulsatile-flow LVAD: group P, n=31; continuous-flow LVAD: group C, n=30). Serial echocardiograms, serum levels of brain natriuretic peptide (BNP), and extracellular matrix biomarkers (ECM) were compared between the groups. Myocardial BNP and ECM gene expression were evaluated in a subset of 18 patients. Postoperative LV ejection fraction was greater (33.2±12.6% versus 17.6±8.8%, P<0.0001) and the mitral E/E′ was lower (9.9±2.6 versus 13.2±3.8, P=0.0002) in group P versus group C. Postoperative serum levels of BNP, metalloproteinases (MMP)-9, and tissue inhibitor of MMP (TIMP)-4 were significantly lower in group P compared with group C (BNP: 552.6±340.6 versus 965.4±805.7 pg/mL, P<0.01; MMP9: 309.0±220.2 versus 475.2±336.9 ng/dL, P<0.05; TIMP4: 1490.9±622.4 versus 2014.3±452.4 ng/dL, P<0.001). Myocardial gene expression of ECM markers and BNP decreased in both groups; however, expression of TIMP-4 decreased only in group P (P=0.024). Conclusions— Mechanical unloading of the failing myocardium using pulsatile devices is more effective as indicated by echocardiographic parameters of systolic and diastolic LV function as well as dynamics of BNP and ECM markers. Therefore, specific effects of pulsatile mechanical unloading on the failing myocardium may have important implications for device selection especially for the purpose of bridge-to-recovery in patients with advanced HF.

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.

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.

Requirement of enhanced Survival Motoneuron protein imposed during neuromuscular junction maturation.

Spinal muscular atrophy is a common motor neuron disease caused by low survival motoneuron (SMN), a key protein in the proper splicing of genes. Restoring the protein is therefore a promising therapeutic strategy. Implementation of this strategy, however, depends on defining the temporal requirements for SMN. Here, we used controlled knockdown of SMN in transgenic mice to determine the precise postnatal stage requirements for this protein. Reducing SMN in neonatal mice resulted in a classic SMA-like phenotype. Unexpectedly, depletion of SMN in adults had relatively little effect. Insensitivity to low SMN emerged abruptly at postnatal day 17, which coincided with establishment of the fully mature neuromuscular junction (NMJ). Mature animals depleted of SMN eventually exhibited evidence of selective neuromuscular pathology that was made worse by traumatic injury. The ability to regenerate the mature NMJ in aged or injured SMN-depleted mice was grossly impaired, a likely consequence of the inability to meet the surge in demand for motoneuronal SMN that was seen in controls. Our results demonstrate that relative maturity of the NMJ determines the temporal requirement for the SMN protein. These observations suggest that the use of potent but potentially deleterious SMN-enhancing agents could be tapered in human patients once the neuromuscular system matures and reintroduced as needed to enhance SMN for remodeling aged or injured NMJs.

Treatment with selumetinib preserves cardiac function and improves survival in cardiomyopathy caused by mutation in the lamin A/C gene

AIMS Mutations in A-type nuclear lamins gene, LMNA, lead to a dilated cardiomyopathy. We have reported abnormal activation of the extracellular signal-regulated kinase1/2 (ERK1/2) signalling in hearts from Lmna(H222P/H222P) mice, which develop dilated cardiomyopathy. We therefore determined whether an inhibitor of ERK1/2 signalling that has been investigated in clinical trials for cancer has the potential to be translated to humans with LMNA cardiomyopathy. METHODS AND RESULTS To evaluate the relevance of this finding in mice to patients, we analysed the ERK1/2 signalling in heart tissue from human subjects with LMNA cardiomyopathy and showed that it was abnormally activated. To determine whether pharmacological inhibitors of the ERK1/2 signalling pathway could potentially be used to treat LMNA cardiomyopathy, we administered selumetinib to male Lmna(H222P/H222P) mice starting at 16 weeks of age, after they show signs of cardiac deterioration, up to 20 weeks of age. Selumetinib is an inhibitor of ERK1/2 signalling and has been given safely to human subjects in clinical trials for cancer. Systemic treatment with selumetinib inhibited cardiac ERK1/2 phosphorylation and blocked increased expression of RNAs encoding natriuretic peptide precursors and proteins involved in sarcomere architecture that occurred in placebo-treated mice. Echocardiography and histological analysis demonstrated that treatment increases cardiac fractional shortening, prevents myocardial fibrosis, and prolongs survival. Selumetinib treatment did not induce biochemical abnormalities suggestive of renal or hepatic toxicity. CONCLUSION Our results suggest that selumetinib or other related inhibitors that have been safely administered to humans in clinical trials could potentially be used to treat LMNA cardiomyopathy.

Aortic Valve Calcification in Mild Primary Hyperparathyroidism

CONTEXT It is unclear whether cardiovascular disease is present in primary hyperparathyroidism (PHPT). OBJECTIVE Aortic valve structure and function were compared in PHPT patients and population-based controls. DESIGN This is a case-control study. SETTING The study was conducted in a university hospital metabolic bone disease unit. PARTICIPANTS We studied 51 patients with PHPT and 49 controls. OUTCOME MEASURES We measured the aortic valve calcification area and the transaortic pressure gradient. RESULTS Aortic valve calcification area was significantly higher in PHPT (0.24 ± 0.02 vs. 0.17 ± 0.02 cm(2), p<0.01), although there was no difference in the peak transaortic pressure gradient, a functional measure of valvular calcification (5.6 ± 0.3 vs. 6.0 ± 0.3 mm Hg, P = 0.39). Aortic valve calcification area was positively associated with PTH (r = 0.34; P < 0.05) but not with serum calcium, phosphorus, or 25-hydroxyvitamin D levels or with calcium-phosphate product. Serum PTH level remained an independent predictor of aortic valve calcification area after adjustment for age, sex, body mass index, smoking status, history of hypercholesterolemia and hypertension, and estimated glomerular filtration rate. CONCLUSIONS Mild PHPT is associated with subclinical aortic valve calcification. PTH, but not serum calcium concentration, predicted aortic valve calcification. PTH was a more important predictor of aortic valve calcification than well-accepted cardiovascular risk factors.

LA volumes and reservoir function are associated with subclinical cerebrovascular disease: the CABL (Cardiovascular Abnormalities and Brain Lesions) study.

OBJECTIVES The purpose of this study was to assess the relationship of left atrial (LA) phasic volumes and LA reservoir function with subclinical cerebrovascular disease in a stroke-free community-based cohort. BACKGROUND An increase in LA size is associated with cardiovascular events including stroke. However, it is not known whether LA phasic volumes and reservoir function are associated with subclinical cerebrovascular disease. METHODS The LA minimum (LAV(min)) and maximum (LAV(max)) volumes, and LA reservoir function, measured as total emptying volume (LAEV) and total emptying fraction (LAEF), were assessed by real-time 3-dimensional echocardiography in 455 stroke-free participants from the community-based CABL (Cardiovascular Abnormalities and Brain Lesions) study. Subclinical cerebrovascular disease was assessed as silent brain infarcts (SBI) and white matter hyperintensity volume (WMHV) by brain magnetic resonance imaging. RESULTS Prevalence of SBI was 15.4%; mean WMHV was 0.66 ± 0.92%. Participants with SBI showed greater LAV(min) (17.1 ± 9.3 ml/m(2) vs. 12.5 ± 5.6 ml/m(2), p < 0.01) and LAV(max) (26.6 ± 8.8 ml/m(2) vs. 23.3 ± 7.0 ml/m(2), p < 0.01) compared to those without SBI. The LAEV (9.5 ± 3.4 ml/m(2) vs. 10.8 ± 3.9 ml/m(2), p < 0.01) and LAEF (38.7 ± 14.7% vs. 47.0 ± 11.9%, p < 0.01) were also reduced in participants with SBI. In univariate analyses, greater LA volumes and smaller reservoir function were significantly associated with greater WMHV. In multivariate analyses, LAV(min) remained significantly associated with SBI (adjusted odds ratio per SD increase: 1.37, 95% confidence interval: 1.04 to 1.80, p < 0.05) and with WMHV (β = 0.12, p < 0.01), whereas LAVmax was not independently associated with either. Smaller LAEF was independently associated with SBI (adjusted odds ratio: 0.67, 95% confidence interval: 0.50 to 0.90, p < 0.01) and WMHV (β = -0.09, p < 0.05). CONCLUSIONS Greater LA volumes and reduced LA reservoir function are associated with subclinical cerebrovascular disease detected by brain magnetic resonance imaging in subjects without history of stroke. In particular, LAV(min) and LAEF are more strongly associated with SBI and WMHV than the more commonly measured LAVmax, and their relationship with subclinical brain lesions is independent of other cardiovascular risk factors.

Depletion of extracellular signal-regulated kinase 1 in mice with cardiomyopathy caused by lamin A/C gene mutation partially prevents pathology before isoenzyme activation

Mutations in the lamin A/C gene (LMNA) encoding A-type nuclear lamins cause dilated cardiomyopathy with variable muscular dystrophy. These mutations enhance mitogen-activated protein kinase signaling in the heart and pharmacological inhibition of extracellular signal-regulated kinase (ERK) 1 and 2 improves cardiac function in Lmna(H222P/H222P) mice. In the current study, we crossed mice lacking ERK1 to Lmna(H222P/H222P) mice and examined cardiac performance and survival. Male Lmna(H222P/H222P)/Erk1(-/-) mice lacking ERK1 had smaller left ventricular end systolic diameters and increased fractional shortening (FS) at 16 weeks of age than Lmna(H222P/H222P/)Erk1(+/+) mice. Their mean survival was also significantly longer. However, the improved cardiac function was abrogated at 20 weeks of age concurrent with an increased activity of ERK2. Lmna(H222P/H222P)/Erk1(-/-) mice treated with an inhibitor of ERK1/2 activation had smaller left ventricular diameters and increased FS at 20 weeks of age. These results provide genetic evidence that ERK1 and ERK2 contribute to the development of cardiomyopathy caused by LMNA mutations and reveal interplay between these isoenzymes in maintaining a combined pathological activity in heart.

Effect of parathyroidectomy on subclinical cardiovascular disease in mild primary hyperparathyroidism

OBJECTIVE We recently demonstrated that mild primary hyperparathyroidism (PHPT) is associated with increased carotid intima-media thickness (IMT) and stiffness, and increased aortic valve calcification. It is unclear whether parathyroidectomy (PTX) improves these abnormalities. The purpose of this study was to determine whether cardiovascular abnormalities in PHPT improve with PTX. DESIGN Forty-four patients with PHPT were studied using carotid ultrasound and transthoracic echocardiography before and after PTX. Carotid IMT, carotid plaque and stiffness, left ventricular mass index (LVMI), myocardial and valvular calcification, and diastolic function were measured before, 1- and 2-year post-PTX. RESULTS Two years after PTX, increased carotid stiffness tended to decline to the normal range (17%, P=0.056) while elevated carotid IMT did not improve. Carotid plaque number and thickness, LVMI and cardiac calcifications did not change after PTX, while some measures of diastolic function (isovolumic relaxation time (IVRT) and tissue Doppler peak early diastolic velocity) worsened within the normal range. Indices did improve in patients with cardiovascular abnormalities at baseline. Increased carotid stiffness improved by 28% (P=0.004), a decline likely to be of clinical significance. More limited improvements also occurred in elevated IMT (3%, P=0.017) and abnormal IVRT (13%, P<0.05), a measure of diastolic dysfunction. CONCLUSIONS In mild PHPT, PTX led to modest changes in some cardiovascular indices. Improvements were mainly evident in those with preexisting cardiovascular abnormalities, particularly elevated carotid stiffness. These findings are reassuring with regard to current international guidelines that do not include cardiovascular disease as a criterion for PTX.