A global in vivo Drosophila RNAi screen identifies NOT3 as a conserved regulator of heart function.

Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.

Direct Reprogramming Improves Cardiac Function and Reverses Fibrosis in Chronic Myocardial Infarction.

BACKGROUND: Because adult cardiomyocytes have little regenerative capacity, resident cardiac fibroblasts (CFs) synthesize extracellular matrix after myocardial infarction (MI) to form fibrosis, leading to cardiac dysfunction and heart failure. Therapies that can regenerate the myocardium and reverse fibrosis in chronic MI are lacking. The overexpression of cardiac transcription factors, including Mef2c/Gata4/Tbx5/Hand2 (MGTH), can directly reprogram CFs into induced cardiomyocytes (iCMs) and improve cardiac function under acute MI. However, the ability of in vivo cardiac reprogramming to repair chronic MI with established scars is undetermined. METHODS: We generated a novel Tcf21iCre/reporter/MGTH2A transgenic mouse system in which tamoxifen treatment could induce both MGTH and reporter expression in the resident CFs for cardiac reprogramming and fibroblast lineage tracing. We first tested the efficacy of this transgenic system in vitro and in vivo for acute MI. Next, we analyzed in vivo cardiac reprogramming and fusion events under chronic MI using Tcf21iCre/Tomato/MGTH2A and Tcf21iCre/mTmG/MGTH2A mice, respectively. Microarray and single-cell RNA sequencing were performed to determine the mechanism of cardiac repair by in vivo reprogramming. RESULTS: We confirmed the efficacy of transgenic in vitro and in vivo cardiac reprogramming for acute MI. In chronic MI, in vivo cardiac reprogramming converted ≈2% of resident CFs into iCMs, in which a majority of iCMs were generated by means of bona fide cardiac reprogramming rather than by fusion with cardiomyocytes. Cardiac reprogramming significantly improved myocardial contraction and reduced fibrosis in chronic MI. Microarray analyses revealed that the overexpression of MGTH activated cardiac program and concomitantly suppressed fibroblast and inflammatory signatures in chronic MI. Single-cell RNA sequencing demonstrated that resident CFs consisted of 7 subclusters, in which the profibrotic CF population increased under chronic MI. Cardiac reprogramming suppressed fibroblastic gene expression in chronic MI by means of conversion of profibrotic CFs to a quiescent antifibrotic state. MGTH overexpression induced antifibrotic effects partly by suppression of Meox1, a central regulator of fibroblast activation. CONCLUSIONS: These results demonstrate that cardiac reprogramming could repair chronic MI by means of myocardial regeneration and reduction of fibrosis. These findings present opportunities for the development of new therapies for chronic MI and heart failure.

Stem Cell Aging in Skeletal Muscle Regeneration and Disease.

Skeletal muscle comprises 30-40% of the weight of a healthy human body and is required for voluntary movements in humans. Mature skeletal muscle is formed by multinuclear cells, which are called myofibers. Formation of myofibers depends on the proliferation, differentiation, and fusion of muscle progenitor cells during development and after injury. Muscle progenitor cells are derived from muscle satellite (stem) cells (MuSCs), which reside on the surface of the myofiber but beneath the basement membrane. MuSCs play a central role in postnatal maintenance, growth, repair, and regeneration of skeletal muscle. In sedentary adult muscle, MuSCs are mitotically quiescent, but are promptly activated in response to muscle injury. Physiological and chronological aging induces MuSC aging, leading to an impaired regenerative capability. Importantly, in pathological situations, repetitive muscle injury induces early impairment of MuSCs due to stem cell aging and leads to early impairment of regeneration ability. In this review, we discuss (1) the role of MuSCs in muscle regeneration, (2) stem cell aging under physiological and pathological conditions, and (3) prospects related to clinical applications of controlling MuSCs.

Enhancement of the Olfactory Response by Lipocalin Cp-Lip1 in Newt Olfactory Receptor Cells: An Electrophysiological Study.

Previously, we have detected the expression of 2 lipocalin genes (lp1 and lp2) in the olfactory epithelium of the Japanese newt Cynops pyrrhogaster. Recombinant proteins of these genes (Cp-Lip1 and Cp-Lip2, respectively) exhibited high affinities to various odorants, suggesting that they work like the odorant-binding proteins (OBPs). However, the physiological functions of OBP generally remain inconclusive. Here, we examined the effect of Cp-Lip1 on the electrophysiological responses of newt olfactory receptor cells. We observed that the electro-olfactogram induced by the vapor of an odorant with high affinity to Cp-Lip1 appeared to increase in amplitude when a tiny drop of Cp-Lip1 solution was dispersed over the olfactory epithelium. However, the analysis was difficult because of possible interference by intrinsic components in the nasal mucus. We subsequently adopted a mucus-free condition by using suction electrode recordings from isolated olfactory cells, in which impulses were generated by puffs of odorant solution. When various concentration (0-5 µM) of Cp-Lip1 was mixed with the stimulus solution of odorants highly affinitive to Cp-Lip1, the impulse frequency increased in a concentration-dependent manner. The increase by Cp-Lip1 was seen more evidently at lower concentration ranges of stimulus odorants. These results strongly suggest that Cp-Lip1 broadens the sensitivity of the olfactory cells toward the lower concentration of odorants, by which animals can detect very low concentration of odorants.

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming.

Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression.

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming.

Induction of human cardiomyocyte-like cells from fibroblasts by defined factors.

Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca(2+) oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2'-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.

Strategies for heart regeneration: approaches ranging from induced pluripotent stem cells to direct cardiac reprogramming.

Cardiovascular disease remains a leading cause of death for which current therapeutic regimens are limited. Following myocardial injury, endogenous cardiac fibroblasts, which account for more than half of the cells in the heart, proliferate and synthesize extracellular matrix, leading to fibrosis and heart failure. As terminally differentiated cardiomyocytes have little regenerative capacity following injury, development of cardiac regenerative therapy is highly desired. Embryonic stem (ES) and induced pluripotent stem (iPS) cells are promising tools for regenerative medicine; however, these stem cells demonstrate variable cardiac differentiation efficiency and tumorigenicity, which should be solved for clinical applications. Up until the last decade, it was an established theory that cardiomyocytes could only be produced from fibroblasts mediating through stem cells. However, in 2010, we reported for the first time a novel method of the direct reprogramming of fibroblasts into cardiomyocytes, demonstrating various reprogramming pathways exist. This review summarizes the latest trends in stem cell and regenerative research, touching upon iPS cells, partial reprogramming strategy, and direct cardiac reprogramming. Specifically, we examine the many recent advances in both in vitro and in vivo direct cardiac reprogramming, and explore the application of these methods to cardiovascular regenerative medicine.

Suppression of Rad leads to arrhythmogenesis via PKA-mediated phosphorylation of ryanodine receptor activity in the heart.

Ras-related small G-protein Rad plays a critical role in generating arrhythmias via regulation of the L-type Ca(2+) channel (LTCC). The aim was to demonstrate the role of Rad in intracellular calcium homeostasis by cardiac-Specific dominant-negative suppression of Rad. Transgenic (TG) mice overexpressing dominant-negative mutant Rad (S105N Rad TG) were generated. To measure intracellular Ca(2+) concentration ([Ca(2+)]i), we recorded [Ca(2+)]i transients and Ca(2+) sparks from isolated cardiomyocytes using confocal microscopy. The mean [Ca(2+)]i transient amplitude was significantly increased in S105N Rad TG cardiomyocytes, compared with control littermate mouse cells. The frequency of Ca(2+) sparks was also significantly higher in TG cells than in control cells, although there were no significant differences in amplitude. The sarcoplasmic reticulum Ca(2+) content was not altered in the S105N Rad TG cells, as assessed by measuring caffeine-induced [Ca(2+)]i transient. In contrast, phosphorylation of Ser(2809) on the cardiac ryanodine receptor (RyR2) was significantly enhanced in TG mouse hearts compared with controls. Additionally, the Rad-mediated RyR2 phosphorylation was regulated via a direct interaction of Rad with protein kinase A (PKA).

Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c, and Tbx5.

RATIONALE: After myocardial infarction (MI), massive cell death in the myocardium initiates fibrosis and scar formation, leading to heart failure. We recently found that a combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), reprograms fibroblasts directly into functional cardiomyocytes in vitro. OBJECTIVE: To investigate whether viral gene transfer of GMT into infarcted hearts induces cardiomyocyte generation. METHODS AND RESULTS: Coronary artery ligation was used to generate MI in the mouse. In vitro transduction of GMT retrovirus converted cardiac fibroblasts from the infarct region into cardiomyocyte-like cells with cardiac-specific gene expression and sarcomeric structures. Injection of the green fluorescent protein (GFP) retrovirus into mouse hearts, immediately after MI, infected only proliferating noncardiomyocytes, mainly fibroblasts, in the infarct region. The GFP expression diminished after 2 weeks in immunocompetent mice but remained stable for 3 months in immunosuppressed mice, in which cardiac induction did not occur. In contrast, injection of GMT retrovirus into α-myosin heavy chain (αMHC)-GFP transgenic mouse hearts induced the expression of αMHC-GFP, a marker of cardiomyocytes, in 3% of virus-infected cells after 1 week. A pooled GMT injection into the immunosuppressed mouse hearts induced cardiac marker expression in retrovirus-infected cells within 2 weeks, although few cells showed striated muscle structures. To transduce GMT efficiently in vivo, we generated a polycistronic retrovirus expressing GMT separated by 2A "self-cleaving" peptides (3F2A). The 3F2A-induced cardiomyocyte-like cells in fibrotic tissue expressed sarcomeric α-actinin and cardiac troponin T and had clear cross striations. Quantitative RT-PCR also demonstrated that FACS-sorted 3F2A-transduced cells expressed cardiac-specific genes. CONCLUSIONS: GMT gene transfer induced cardiomyocyte-like cells in infarcted hearts.

Nongenetic method for purifying stem cell-derived cardiomyocytes.

Several applications of pluripotent stem cell (PSC)-derived cardiomyocytes require elimination of undifferentiated cells. A major limitation for cardiomyocyte purification is the lack of easy and specific cell marking techniques. We found that a fluorescent dye that labels mitochondria, tetramethylrhodamine methyl ester perchlorate, could be used to selectively mark embryonic and neonatal rat cardiomyocytes, as well as mouse, marmoset and human PSC-derived cardiomyocytes, and that the cells could subsequently be enriched (>99% purity) by fluorescence-activated cell sorting. Purified cardiomyocytes transplanted into testes did not induce teratoma formation. Moreover, aggregate formation of PSC-derived cardiomyocytes through homophilic cell-cell adhesion improved their survival in the immunodeficient mouse heart. Our approaches will aid in the future success of using PSC-derived cardiomyocytes for basic and clinical applications.

Associations between glycemic variability, sleep quality, and daily steps in subjects without diabetes using wearable devices.

Background: Since there are limited studies on the associations between glycemic variability (GV) and sleep quality or physical activity in subjects without diabetes, we evaluated the associations between GV, as assessed by continuous glucose monitoring (CGM), and both sleep quality and daily steps using wearable devices in healthy individuals. Methods: Forty participants without diabetes were monitored by both an intermittently scanned CGM and a smartwatch-type activity tracker for 2 weeks. The standard deviation (SD) and coefficient of variation (CV) of glucose were evaluated as indices of GV. The activity tracker was used to calculate each participant's average step count per day. We also calculated sleep duration, sleep efficiency, and sleep latency based on data from the activity tracker. Spearman's correlation coefficient was used to assess the association between GV and sleep indices or daily steps. For each participant, periods were divided into quartiles according to step counts throughout the day. We compared mean parameter differences between the periods of lowest quartile and highest quartile (lower 25% and upper 25%). Results: SD glucose was significantly positively correlated with sleep latency (R = 0.23, P < 0.05). There were no significant correlations among other indices in GV and sleep quality (P > 0.05). SD glucose and CV glucose levels in the upper 25% period of daily steps were lower than those in the lower 25% period in each participant (both, P < 0.01). Conclusion: In subjects without diabetes, GV evaluated by intermittently scanned CGM was positively associated with the time to fall asleep. Furthermore, GV in the days of larger daily steps was decreased compared to the days of smaller daily steps in each participant.

Assessment of glycemic variability and lifestyle behaviors in healthy nondiabetic individuals according to the categories of body mass index.

BACKGROUND: There are limited data about the association between body mass index (BMI), glycemic variability (GV), and life-related factors in healthy nondiabetic adults. METHODS: This cross-sectional study was carried out within our ethics committee-approved study called "Exploring the impact of nutrition advice on blood sugar and psychological status using continuous glucose monitoring (CGM) and wearable devices". Prediabetes was defined by the HbA1c level of 5.7-6.4% and /or fasting glucose level of 100-125 mg/dL. Glucose levels and daily steps were measured for 40 participants using Free Style Libre and Fitbit Inspire 2 under normal conditions for 14 days. Dietary intakes and eating behaviors were assessed using a brief-type self-administered dietary history questionnaire and a modified questionnaire from the Obesity Guidelines. RESULTS: All indices of GV were higher in the prediabetes group than in the healthy group, but a significant difference was observed only in mean amplitude of glycemic excursions (MAGE). In the multivariate analysis, only the presence of prediabetes showed a significant association with the risk of higher than median MAGE (Odds, 6.786; 95% CI, 1.596-28.858; P = 0.010). Additionally, the underweight (BMI < 18.5) group had significantly higher value in standard deviation (23.7 ± 3.5 vs 19.8 ± 3.7 mg/dL, P = 0.038) and coefficient variability (22.6 ± 4.6 vs 18.4 ± 3.2%, P = 0.015), compared to the normal group. This GV can be partially attributed to irregularity of eating habits. On the contrary, the overweight (BMI ≥ 25) group had the longest time above the 140 or 180 mg/dL range, which may be due to eating style and taking fewer steps (6394 ± 2337 vs 9749 ± 2408 steps, P = 0.013). CONCLUSIONS: Concurrent CGM with diet and activity monitoring could reduce postprandial hyperglycemia through assessment of diet and daily activity, especially in non- normal weight individuals.

MEF2C/p300-mediated epigenetic remodeling promotes the maturation of induced cardiomyocytes.

Cardiac transcription factors (TFs) directly reprogram fibroblasts into induced cardiomyocytes (iCMs), where MEF2C acts as a pioneer factor with GATA4 and TBX5 (GT). However, the generation of functional and mature iCMs is inefficient, and the molecular mechanisms underlying this process remain largely unknown. Here, we found that the overexpression of transcriptionally activated MEF2C via fusion of the powerful MYOD transactivation domain combined with GT increased the generation of beating iCMs by 30-fold. Activated MEF2C with GT generated iCMs that were transcriptionally, structurally, and functionally more mature than those generated by native MEF2C with GT. Mechanistically, activated MEF2C recruited p300 and multiple cardiogenic TFs to cardiac loci to induce chromatin remodeling. In contrast, p300 inhibition suppressed cardiac gene expression, inhibited iCM maturation, and decreased the beating iCM numbers. Splicing isoforms of MEF2C with similar transcriptional activities did not promote functional iCM generation. Thus, MEF2C/p300-mediated epigenetic remodeling promotes iCM maturation.

Fabry disease associated with multiple myeloma: a case report.

Fabry disease (FD) is an X-linked genetic lysosomal disorder caused by alpha-galactosidase A (GLA) deficiency. Multiple myeloma (MM) predominately affects older adults, which ranks as the second commonest hematological malignancy. Their overlap has rarely been reported. We present a case of the coexistence of FD and MM in a patient. We report the case of a 68-year-old woman who was referred to our hospital for the evaluation of thoracic spine tumor with bone destruction. On admission, her serum creatinine (Cr) level was elevated to 12.70 mg/dL from the baseline value of 0.91 mg/dL. Bone marrow aspiration revealed MM. Renal biopsy showed myeloma cast nephropathy, which was the primary cause of acute kidney injury. Renal pathology also showed podocyte swelling and tubule myeloid bodies in a mosaic pattern compatible with female FD. Consequently, the patient was diagnosed as FD based on the germ line mutation in GLA. The patient was treated with bortezomib and dexamethasone therapy, which significantly improved the renal function. This is the second case demonstrating a potential pathogenic relationship between FD and MM. Since FD is one of the few genetic diseases for which there are therapeutic agents with fewer side effects, diagnostic value of FD is high. If an MM patient has multiple organ abnormalities or any familial history, the physician should suspect FD.

Upregulation of neuropeptide Y in cardiac sympathetic nerves induces stress (Takotsubo) cardiomyopathy.

Substantial emotional or physical stress may lead to an imbalance in the brain, resulting in stress cardiomyopathy (SC) and transient left ventricular (LV) apical ballooning. Even though these conditions are severe, their precise underlying mechanisms remain unclear. Appropriate animal models are needed to elucidate the precise mechanisms. In this study, we established a new animal model of epilepsy-induced SC. The SC model showed an increased expression of the acute phase reaction protein, c-Fos, in the paraventricular hypothalamic nucleus (PVN), which is the sympathetic nerve center of the brain. Furthermore, we observed a significant upregulation of neuropeptide Y (NPY) expression in the left stellate ganglion (SG) and cardiac sympathetic nerves. NPY showed neither positive nor negative inotropic and chronotropic effects. On the contrary, NPY could interrupt ß-adrenergic signaling in cardiomyocytes when exposure to NPY precedes exposure to noradrenaline. Moreover, its elimination in the left SG via siRNA treatment tended to reduce the incidence of SC. Thus, our results indicated that upstream sympathetic activation induced significant upregulation of NPY in the left SG and cardiac sympathetic nerves, resulting in cardiac dysfunctions like SC.

The complement C3-complement factor D-C3a receptor signalling axis regulates cardiac remodelling in right ventricular failure.

Failure of the right ventricle plays a critical role in any type of heart failure. However, the mechanism remains unclear, and there is no specific therapy. Here, we show that the right ventricle predominantly expresses alternative complement pathway-related genes, including Cfd and C3aR1. Complement 3 (C3)-knockout attenuates right ventricular dysfunction and fibrosis in a mouse model of right ventricular failure. C3a is produced from C3 by the C3 convertase complex, which includes the essential component complement factor D (Cfd). Cfd-knockout mice also show attenuation of right ventricular failure. Moreover, the plasma concentration of CFD correlates with the severity of right ventricular failure in patients with chronic right ventricular failure. A C3a receptor (C3aR) antagonist dramatically improves right ventricular dysfunction in mice. In summary, we demonstrate the crucial role of the C3-Cfd-C3aR axis in right ventricular failure and highlight potential therapeutic targets for right ventricular failure.

Impact of long-term caloric restriction on cardiac senescence: caloric restriction ameliorates cardiac diastolic dysfunction associated with aging.

Approximately half of older patients with congestive heart failure have normal left ventricular (LV) systolic but abnormal LV diastolic function. In mammalian hearts, aging is associated with LV diastolic dysfunction. Caloric restriction (CR) is expected to retard cellular senescence and to attenuate the physiological decline in organ function. Therefore, the aim of the present study was to investigate the impact of long-term CR on cardiac senescence, in particular the effect of CR on LV diastolic dysfunction associated with aging. Male 8-month-old Fischer344 rats were divided into ad libitum fed and CR (40% energy reduction) groups. LV function was evaluated by echocardiography and cardiac senescence was compared between the two groups at the age of 30-month-old. (1) Echocardiography showed similar LV systolic function, but better LV diastolic function in the CR group. (2) Histological analysis revealed that CR attenuated the accumulation of senescence-associated ß-galactosidase and lipofuscin and reduced myocyte apoptosis. (3) In measurements of [Ca(2+)](i) transients, the time to 50% relaxation was significantly smaller in the CR group, whereas F/F(0) was similar. (4) CR attenuated the decrease in sarcoplasmic reticulum calcium ATPase 2 protein with aging. (5) CR suppressed the mammalian target of rapamycin (mTOR) pathway and increased the ratio of conjugated to cytosolic light chain 3, suggesting that autophagy is enhanced in the CR hearts. In conclusion, CR improves diastolic function in the senescent myocardium by amelioration of the age-associated deterioration in intracellular Ca(2+) handling. Enhanced autophagy via the suppression of mTOR during CR may retard cardiac senescence.

Cardiac regeneration by direct reprogramming in this decade and beyond.

Japan faces an increasing incidence of heart disease, owing to a shift towards a westernized lifestyle and an aging demographic. In cases where conventional interventions are not appropriate, regenerative medicine offers a promising therapeutic option. However, the use of stem cells has limitations, and therefore, "direct cardiac reprogramming" is emerging as an alternative treatment. Myocardial regeneration transdifferentiates cardiac fibroblasts into cardiomyocytes in situ.Three cardiogenic transcription factors: Gata4, Mef2c, and Tbx5 (GMT) can induce direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs), in mice. However, in humans, additional factors, such as Mesp1 and Myocd, are required. Inflammation and immune responses hinder the reprogramming process in mice, and epigenetic modifiers such as TET1 are involved in direct cardiac reprogramming in humans. The three main approaches to improving reprogramming efficiency are (1) improving direct cardiac reprogramming factors, (2) improving cell culture conditions, and (3) regulating epigenetic factors. miR-133 is a potential candidate for the first approach. For the second approach, inhibitors of TGF-ß and Wnt signals, Akt1 overexpression, Notch signaling pathway inhibitors, such as DAPT ((S)-tert-butyl 2-((S)-2-(2-(3,5-difluorophenyl) acetamido) propanamido)-2-phenylacetate), fibroblast growth factor (FGF)-2, FGF-10, and vascular endothelial growth factor (VEGF: FFV) can influence reprogramming. Reducing the expression of Bmi1, which regulates the mono-ubiquitination of histone H2A, alters histone modification, and subsequently the reprogramming efficiency, in the third approach. In addition, diclofenac, a non-steroidal anti-inflammatory drug, and high level of Mef2c overexpression could improve direct cardiac reprogramming.Direct cardiac reprogramming needs improvement if it is to be used in humans, and the molecular mechanisms involved remain largely elusive. Further advances in cardiac reprogramming research are needed to bring us closer to cardiac regenerative therapy.