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CD36 is one of the important transporters of long-chain fatty acids (LCFAs) in the myocardium. We previously reported that CD36-deficient patients demonstrate a marked reduction of myocardial uptake of LCFA, while myocardial glucose uptake shows a compensatory increase, and are often accompanied by cardiomyopathy. However, the molecular mechanisms and functional role of CD36 in the myocardium remain unknown.The current study aimed to explore the pathophysiological role of CD36 in the heart. Methods: Using wild type (WT) and knockout (KO) mice, we generated pressure overload by transverse aortic constriction (TAC) and analyzed cardiac functions by echocardiography. To assess cardiac hypertrophy and fibrosis, histological and molecular analyses and measurement of ATP concentration in mouse hearts were performed.By applying TAC, the survival rate was significantly lower in KO than that in WT mice. After TAC, KO mice showed significantly higher heart weight-to-tibial length ratio and larger cross-sectional area of cardiomyocytes than those of WT. Although left ventricular (LV) wall thickness in the KO mice was similar to that in the WT mice, the KO mice showed a significant enlargement of LV cavity and reduced LV fractional shortening compared to the WT mice with TAC. A tendency for decreased myocardial ATP concentration was observed in the KO mice compared to the WT mice after TAC operation.These data suggest that the LCFA transporter CD36 is required for the maintenance of energy provision, systolic function, and myocardial structure.

Pressure overload induces a transition from cardiac hypertrophy to heart failure, but its underlying mechanisms remain elusive. Here we reconstruct a trajectory of cardiomyocyte remodeling and clarify distinct cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure, by integrating single-cardiomyocyte transcriptome with cell morphology, epigenomic state and heart function. During early hypertrophy, cardiomyocytes activate mitochondrial translation/metabolism genes, whose expression is correlated with cell size and linked to ERK1/2 and NRF1/2 transcriptional networks. Persistent overload leads to a bifurcation into adaptive and failing cardiomyocytes, and p53 signaling is specifically activated in late hypertrophy. Cardiomyocyte-specific p53 deletion shows that cardiomyocyte remodeling is initiated by p53-independent mitochondrial activation and morphological hypertrophy, followed by p53-dependent mitochondrial inhibition, morphological elongation, and heart failure gene program activation. Human single-cardiomyocyte analysis validates the conservation of the pathogenic transcriptional signatures. Collectively, cardiomyocyte identity is encoded in transcriptional programs that orchestrate morphological and functional phenotypes.

The DNA damage response (DDR) plays a pivotal role in maintaining genome integrity. DNA damage and DDR activation are observed in the failing heart, however, the type of DNA damage and its role in the pathogenesis of heart failure remain elusive. Here we show the critical role of DNA single-strand break (SSB) in the pathogenesis of pressure overload-induced heart failure. Accumulation of unrepaired SSB is observed in cardiomyocytes of the failing heart. Unrepaired SSB activates DDR and increases the expression of inflammatory cytokines through NF-κB signalling. Pressure overload-induced heart failure is more severe in the mice lacking XRCC1, an essential protein for SSB repair, which is rescued by blocking DDR activation through genetic deletion of ATM, suggesting the causative role of SSB accumulation and DDR activation in the pathogenesis of heart failure. Prevention of SSB accumulation or persistent DDR activation may become a new therapeutic strategy against heart failure.

Cardiac stem cells or precursor cells regenerate cardiomyocytes; however, the mechanism underlying this effect remains unclear. We generated CreLacZ mice in which more than 99.9% of the cardiomyocytes in the left ventricular field were positive for 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) staining immediately after tamoxifen injection. Three months after myocardial infarction (MI), the MI mice had more X-gal-negative (newly generated) cells than the control mice (3.04 ± 0.38/mm2, MI; 0.47 ± 0.16/mm2, sham; p < 0.05). The cardiac side population (CSP) cell fraction contained label-retaining cells, which differentiated into X-gal-negative cardiomyocytes after MI. We injected a leukemia inhibitory factor (LIF)-expression construct at the time of MI and identified a significant functional improvement in the LIF-treated group. At 1 month after MI, in the MI border and scar area, the LIF-injected mice had 31.41 ± 5.83 X-gal-negative cardiomyocytes/mm2, whereas the control mice had 12.34 ± 2.56 X-gal-negative cardiomyocytes/mm2 (p < 0.05). Using 5-ethynyl-2'-deoxyurinide (EdU) administration after MI, the percentages of EdU-positive CSP cells in the LIF-treated and control mice were 29.4 ± 2.7% and 10.6 ± 3.7%, respectively, which suggests that LIF influenced CSP proliferation. Moreover, LIF activated the Janus kinase (JAK)signal transducer and activator of transcription (STAT), mitogen-activated protein kinase/extracellular signal-regulated (MEK)extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)-AKT pathways in CSPs in vivo and in vitro. The enhanced green fluorescent protein (EGFP)-bone marrow-chimeric CreLacZ mouse results indicated that LIF did not stimulate cardiogenesis via circulating bone marrow-derived cells during the 4 weeks following MI. Thus, LIF stimulates, in part, stem cell-derived cardiomyocyte regeneration by activating cardiac stem or precursor cells. This approach may represent a novel therapeutic strategy for cardiogenesis.

During embryogenesis, progenitor cells are specified and differentiated into mature cardiomyocytes. Soon after birth, the ability of cardiomyocytes to proliferate is strongly restrained, and thereafter, they grow in size without cell division. Under pathological conditions, cardiomyocytes show adaptive and maladaptive responses through complex intracellular signaling pathways and cross-talking networks of intercellular and inter-tissue communications, but ultimately, they become dysfunctional and undergo cell death or degeneration. Cardiovascular diseases remain the most prevalent, costly, disabling, and deadly medical conditions. To develop novel therapies for them, it is important to elucidate the underlying mechanisms that govern gene expression, signal transduction to cellular communication. In this review article for the 2014 SATO Memorial Award, an approach to uncover molecular and cellular pathophysiology is summarized, focusing on homeobox transcription factor Nkx2-5 in the transcriptional regulation of the cardiac gene program, 3-phosphoinositide-dependent kinase-1, in the regulation of postnatal cardiomyocyte growth, survival, and function, angiotensin II type 1 receptor in the development of pathological hypertrophy and remodeling, and mast cell infiltration in the pathogenesis of atrial remodeling and fibrillation.

Cardiac hypertrophy is an adaptive response to physiological and pathological overload. In response to the overload, individual cardiac myocytes become mechanically stretched and activate intracellular hypertrophic signaling pathways to re-use embryonic transcription factors and to increase the synthesis of various proteins, such as structural and contractile proteins. These hypertrophic responses increase oxygen demand and promote myocardial angiogenesis to dissolve the hypoxic situation and to maintain cardiac contractile function; thus, these responses suggest crosstalk between cardiac myocytes and microvasculature. However, sustained pathological overload induces maladaptation and cardiac remodeling, resulting in heart failure. In recent years, specific understanding has increased with regard to the molecular processes and cell-cell interactions that coordinate myocardial growth and angiogenesis. In this review, we summarize recent advances in understanding the regulatory mechanisms of coordinated myocardial growth and angiogenesis in the pathophysiology of cardiac hypertrophy and heart failure.

Rationale: The number of patients with coronary heart disease, including myocardial infarction, is increasing and novel therapeutic strategy is awaited. Tumor suppressor protein p53 accumulates in the myocardium after myocardial infarction, causes apoptosis of cardiomyocytes, and plays an important role in the progression into heart failure., Objectives: We investigated the molecular mechanisms of p53 accumulation in the heart after myocardial infarction and tested whether anti-p53 approach would be effective against myocardial infarction., Methods and Results: Through expression screening, we found that CHIP (carboxyl terminus of Hsp70-interacting protein) is an endogenous p53 antagonist in the heart. CHIP suppressed p53 level by ubiquitinating and inducing proteasomal degradation. CHIP transcription was downregulated after hypoxic stress and restoration of CHIP protein level prevented p53 accumulation after hypoxic stress. CHIP overexpression in vivo prevented p53 accumulation and cardiomyocyte apoptosis after myocardial infarction. Promotion of CHIP function by heat shock protein (Hsp)90 inhibitor, 17-allylamino-17-demethoxy geldanamycin (17-AAG), also prevented p53 accumulation and cardiomyocyte apoptosis both in vitro and in vivo. CHIP-mediated p53 degradation was at least one of the cardioprotective effects of 17-AAG., Conclusions: We found that downregulation of CHIP level by hypoxia was responsible for p53 accumulation in the heart after myocardial infarction. Decreasing the amount of p53 prevented myocardial apoptosis and ameliorated ventricular remodeling after myocardial infarction. We conclude that anti-p53 approach would be effective to treat myocardial infarction.

Atrial fibrillation (AF) is a common arrhythmia that increases the risk of stroke and heart failure. Here, we have shown that mast cells, key mediators of allergic and immune responses, are critically involved in AF pathogenesis in stressed mouse hearts. Pressure overload induced mast cell infiltration and fibrosis in the atrium and enhanced AF susceptibility following atrial burst stimulation. Both atrial fibrosis and AF inducibility were attenuated by stabilization of mast cells with cromolyn and by BM reconstitution from mast cell-deficient WBB6F1-KitW/W-v mice. When cocultured with cardiac myocytes or fibroblasts, BM-derived mouse mast cells increased platelet-derived growth factor A (PDGF-A) synthesis and promoted cell proliferation and collagen expression in cardiac fibroblasts. These changes were abolished by treatment with a neutralizing antibody specific for PDGF alpha-receptor (PDGFR-alpha). Consistent with these data, upregulation of atrial Pdgfa expression in pressure-overloaded hearts was suppressed by BM reconstitution from WBB6F1-KitW/W-v mice. Furthermore, injection of the neutralizing PDGFR-alpha-specific antibody attenuated atrial fibrosis and AF inducibility in pressure-overloaded hearts, whereas administration of homodimer of PDGF-A (PDGF-AA) promoted atrial fibrosis and enhanced AF susceptibility in normal hearts. Our results suggest a crucial role for mast cells in AF and highlight a potential application of controlling the mast cell/PDGF-A axis to achieve upstream prevention of AF in stressed hearts.

Type 1 angiotensin II (AT(1)) receptor has a critical role in the development of load-induced cardiac hypertrophy. Recently, we showed that mechanical stretching of cells activates the AT(1) receptor without the involvement of angiotensin II (AngII) and that this AngII-independent activation is inhibited by the inverse agonistic activity of the AT(1) receptor blocker (ARB), candesartan. Although the inverse agonist activity of ARBs has been studied in terms of their action on constitutively active AT(1) receptors, the structure-function relationship of the inverse agonism they exert against stretch-induced AT(1) receptor activation has not been fully elucidated. Assays evaluating c-fos gene expression and phosphorylated extracellular signal-regulated protein kinases (ERKs) have shown that olmesartan has strong inverse agonist activities against the constitutively active AT(1) receptor and the stretch-induced activation of AT(1) receptor, respectively. Ternary drug-receptor interactions, which occur between the hydroxyl group of olmesartan and Tyr(113) and between the carboxyl group of olmesartan and Lys(199) and His(256), were essential for the potent inverse agonist action olmesartan exerts against stretch-induced ERK activation and the constitutive activity of the AT(1)-N111G mutant receptor. Furthermore, the inverse agonist activity olmesartan exerts against stretch-induced ERK activation requires an additional drug-receptor interaction involving the tetrazole group of olmesartan and Gln(257) of the AT(1) receptor. These results suggest that multivalent interactions between an inverse agonist and the AT(1) receptor are required to stabilize the receptor in an inactive conformation in response to the distinct processes that lead to an AngII-independent activation of the AT(1) receptor.

Side population (SP) cells, which can be identified by their ability to exclude Hoechst 33342 dye, are one of the candidates for somatic stem cells. Although bone marrow SP cells are known to be long-term repopulating hematopoietic stem cells, there is little information about the characteristics of cardiac SP cells (CSPs). When cultured CSPs from neonatal rat hearts were treated with oxytocin or trichostatin A, some CSPs expressed cardiac-specific genes and proteins and showed spontaneous beating. When green fluorescent protein-positive CSPs were intravenously infused into adult rats, many more ( approximately 12-fold) CSPs were migrated and homed in injured heart than in normal heart. CSPs in injured heart differentiated into cardiomyocytes, endothelial cells, or smooth muscle cells (4.4%, 6.7%, and 29% of total CSP-derived cells, respectively). These results suggest that CSPs are intrinsic cardiac stem cells and involved in the regeneration of diseased hearts.

Although Wingless (Wg)/Wnt signaling has been implicated in heart development of multiple organisms, conflicting results have been reported regarding the role of Wnt/beta-catenin pathway in cardiac myogenesis: Wg/armadillo signaling promotes heart development in Drosophila, whereas activation of Wnt/beta-catenin signaling inhibits heart formation in avians and amphibians. Using an in vitro system of mouse ES cell differentiation into cardiomyocytes, we show here that Wnt/beta-catenin signaling exhibits developmental stage-specific, biphasic, and antagonistic effects on cardiomyogenesis and hematopoiesis/vasculogenesis. Activation of the Wnt/beta-catenin pathway in the early phase during embryoid body (EB) formation enhances ES cell differentiation into cardiomyocytes while suppressing the differentiation into hematopoietic and vascular cell lineages. In contrast, activation of Wnt/beta-catenin signaling in the late phase after EB formation inhibits cardiomyocyte differentiation and enhances the expression of hematopoietic/vascular marker genes through suppression of bone morphogenetic protein signaling. Thus, Wnt/beta-catenin signaling exhibits biphasic and antagonistic effects on cardiomyogenesis and hematopoiesis/vasculogenesis, depending on the stage of development.

We have recently reported that activation of phosphatidylinositol 3-kinase (PI3K) plays a critical role in the early stage of cardiomyocyte differentiation of P19CL6 cells. We here examined molecular mechanisms of how PI3K is involved in cardiomyocyte differentiation. DNA chip analysis revealed that expression levels of Wnt-3a were markedly increased and that the Wnt/beta-catenin pathway was activated temporally during the early stage of cardiomyocyte differentiation of P19CL6 cells. Activation of the Wnt/beta-catenin pathway during this period was required and sufficient for cardiomyocyte differentiation of P19CL6 cells. Inhibition of the PI3K/Akt pathway suppressed the Wnt/beta-catenin pathway by activation of glycogen synthase kinase-3beta (GSK-3beta) and degradation of beta-catenin. Suppression of cardiomyocyte differentiation by inhibiting the PI3K/Akt pathway was rescued by forced expression of a nonphosphorylated, constitutively active form of beta-catenin. These results suggest that the PI3K pathway regulates cardiomyocyte differentiation through suppressing the GSK-3beta activity and maintaining the Wnt/beta-catenin activity.

Granulocyte colony-stimulating factor (G-CSF) was reported to induce myocardial regeneration by promoting mobilization of bone marrow stem cells to the injured heart after myocardial infarction, but the precise mechanisms of the beneficial effects of G-CSF are not fully understood. Here we show that G-CSF acts directly on cardiomyocytes and promotes their survival after myocardial infarction. G-CSF receptor was expressed on cardiomyocytes and G-CSF activated the Jak/Stat pathway in cardiomyocytes. The G-CSF treatment did not affect initial infarct size at 3 d but improved cardiac function as early as 1 week after myocardial infarction. Moreover, the beneficial effects of G-CSF on cardiac function were reduced by delayed start of the treatment. G-CSF induced antiapoptotic proteins and inhibited apoptotic death of cardiomyocytes in the infarcted hearts. G-CSF also reduced apoptosis of endothelial cells and increased vascularization in the infarcted hearts, further protecting against ischemic injury. All these effects of G-CSF on infarcted hearts were abolished by overexpression of a dominant-negative mutant Stat3 protein in cardiomyocytes. These results suggest that G-CSF promotes survival of cardiac myocytes and prevents left ventricular remodeling after myocardial infarction through the functional communication between cardiomyocytes and noncardiomyocytes.

The concept of the plasticity or transdifferentiation of adult stem cells has been challenged by the phenomenon of cell fusion. In this work, we examined whether neonatal cardiomyocytes fuse with various somatic cells including endothelial cells, cardiac fibroblasts, bone marrow cells, and endothelial progenitor cells spontaneously in vitro. When cardiomyocytes were cocultured with endothelial cells or cardiac fibroblasts, they fused and showed phenotypes of cardiomyocytes. Furthermore, cardiomyocytes reentered the G2-M phase in the cell cycle after fusing with proliferative noncardiomyocytes. Transplanted endothelial cells or skeletal muscle-derived cells fused with adult cardiomyocytes in vivo. In the cryoinjured heart, there were Ki67-positive cells that expressed both cardiac and endothelial lineage marker proteins. These results suggest that cardiomyocytes fuse with other cells and enter the cell cycle by maintaining their phenotypes.

It is still not clear whether loss of cardiomyocytes through programmed cell death causes heart failure. To clarify the role of cell death in heart failure, we generated transgenic mice (TG) that express human diphtheria toxin receptor in the hearts. A mosaic expression pattern of the transgene was observed, and the transgene-expressing cardiomyocytes (17.3% of the total cardiomyocytes) were diffusely scattered throughout the ventricles. Intramuscular injection of diphtheria toxin induced complete elimination of the transgene-expressing cardiomyocytes within 7 days, and approximately 80% of TG showed pathophysiological features characteristic of heart failure and were dead within 14 days. Degenerated cardiomyocytes of the TG heart showed characteristic features indicative of autophagic cell death such as up-regulated lysosomal markers and abundant autophagosomes containing cytosolic organelles like cardiomyocytes of human dilated cardiomyopathy. The heart failure-inducible TG are a useful model for dilated cardiomyopathy, and provided evidence indicating that myocardial cell loss through autophagic cell death plays of a causal role in the pathogenesis heart failure., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

The angiotensin II type 1 (AT1) receptor has a crucial role in load-induced cardiac hypertrophy. Here we show that the AT1 receptor can be activated by mechanical stress through an angiotensin-II-independent mechanism. Without the involvement of angiotensin II, mechanical stress not only activates extracellular-signal-regulated kinases and increases phosphoinositide production in vitro, but also induces cardiac hypertrophy in vivo. Mechanical stretch induces association of the AT1 receptor with Janus kinase 2, and translocation of G proteins into the cytosol. All of these events are inhibited by the AT1 receptor blocker candesartan. Thus, mechanical stress activates AT1 receptor independently of angiotensin II, and this activation can be inhibited by an inverse agonist of the AT1 receptor.

The cardiac homeobox transcription factor CSX/NKX2-5 plays an important role in vertebrate heart development. Using a yeast two-hybrid screening, we identified a novel LIM domain-containing protein, named CSX-associated LIM protein (Cal), that interacts with CSX/NKX2-5. CSX/NKX2-5 and Cal associate with each other both in vivo and in vitro, and the LIM domains of Cal and the homeodomain of CSX/NKX2-5 were necessary for mutual binding. Cal itself possessed the transcription-promoting activity, and cotransfection of Cal enhanced CSX/NKX2-5-induced activation of atrial natriuretic peptide gene promoter. Cal contained a functional nuclear export signal and shuttled from the cytoplasm into the nucleus in response to calcium. Accumulation of Cal in the nucleus of P19CL6 cells promoted myocardial cell differentiation accompanied by increased expression levels of the target genes of CSX/NKX2-5. These results suggest that a novel LIM protein Cal induces cardiomyocyte differentiation through its dynamic intracellular shuttling and association with CSX/NKX2-5., (Copyright The Rockefeller University Press)

Background: Because cardiomyocyte death causes heart failure, it is important to find the molecules that protect cardiomyocytes from death. The death trap is a useful method to identify cell-protective genes., Methods and Results: In this study, we isolated the heat shock transcription factor 1 (HSF1) as a protective molecule by the death trap method. Cell death induced by hydrogen peroxide was prevented by overexpression of HSF1 in COS7 cells. Thermal preconditioning at 42 degrees C for 60 minutes activated HSF1, which played a critical role in survival of cardiomyocytes from oxidative stress. In the heart of transgenic mice overexpressing a constitutively active form of HSF1, ischemia followed by reperfusion-induced ST-segment elevation in ECG was recovered faster, infarct size was smaller, and cardiomyocyte death was less than wild-type mice. Protein kinase B/Akt was more strongly activated, whereas Jun N-terminal kinase and caspase 3 were less activated in transgenic hearts than wild-type ones., Conclusions: These results suggest that HSF1 protects cardiomyocytes from death at least in part through activation of Akt and inactivation of Jun N-terminal kinase and caspase 3.

Reactive oxygen species (ROS) are proposed to contribute to the deterioration of cardiac function in patients with heart diseases. It has been reported that ROS are increased in the failing heart and involved in atherosclerosis, myocardial ischemia/reperfusion injury, and heart failure. Antioxidant enzymes are decreased in the decompensated heart, depressing defense mechanisms against oxidative stress. A variety of proteins, including receptors, ionic channels, transporters, and components of signal transduction pathways, are substrates of oxidation by ROS. ROS also function as signal transduction intermediates to induce transcription factor activation, gene expression, cell growth, and apoptosis. Recently, the upstream and downstream molecules of ROS in signal transduction pathways have been the subjects of intense investigation. These molecules include the mitogen-activated protein kinase family, the Rho family of small GTP binding proteins, the Src family of tyrosine kinases, Ras, and cytokines. The modulation of oxidative stress-induced signaling pathways is effective for preventing the progression of heart diseases.

Background: Myocardial infarction (MI) is a leading cause of cardiac morbidity and mortality in many countries; however, the treatment of MI is still limited., Methods and Results: We demonstrate a novel gene therapy for MI using leukemia inhibitory factor (LIF) cDNA. We injected LIF plasmid DNA into the thigh muscle of mice immediately after inducing MI. Intramuscular injection of LIF cDNA resulted in a marked increase in circulating LIF protein concentrations. Two weeks later, left ventricular remodeling, such as infarct extent and myocardial fibrosis, was markedly attenuated in the LIF cDNA-injected mice compared with vehicle-injected mice. More myocardium was preserved and cardiac function was better in the LIF-treated mice than in the vehicle-injected mice. Injection of LIF cDNA not only prevented the death of cardiomyocytes in the ischemic area but also induced neovascularization in the myocardium. Furthermore, LIF cDNA injection increased the number of cardiomyocytes in cell cycle and enhanced mobilization of bone marrow cells to the heart and their differentiation into cardiomyocytes., Conclusions: The intramuscular injection of LIF cDNA may induce regeneration of myocardium and provide a novel treatment for MI.

Cell transplantation could be a potential therapy for heart damage. Skeletal myoblasts have been expected to be a good cell source for autologous transplantation; however, the safety and efficacy of their transplantation are still controversial. Recent studies have revealed that skeletal muscle possesses the stem cell population that is distinct from myoblasts. To elucidate whether skeletal muscle stem cells can transdifferentiate into cardiomyocytes, we cocultured skeletal muscle cells isolated from transgenic mice expressing green fluorescent protein with cardiomyocytes of neonatal rats. Skeletal muscle-derived cells expressed cardiac-specific proteins such as cardiac troponin T and atrial natriuretic peptide as well as cardiac-enriched transcription factors such as Nkx2E (formerly called Csx/Nkx2.5) and GATA4 by coculture with cardiomyocytes. Skeletal muscle-derived cells also expressed cadherin and connexin 43 at the junctions with neighboring cardiomyocytes. Cardiomyocyte-like action potentials were recorded from beating skeletal muscle-derived cells. Treatment of nifedipine or culture in Ca2+-free media suppressed contraction of cardiomyocytes and inhibited skeletal muscle cells to express cardiac-specific proteins. Cyclic stretch completely restored this inhibitory effect. These results suggest that some part of skeletal muscle cells can transdifferentiate into cardiomyocytes and that direct cell-to-cell contact and contraction of neighboring cardiomyocytes are important for the transdifferentiation.