Do binucleate cardiomyocytes have a role in myocardial repair? Insights using isolated rodent myocytes and cell culture.

Neonatal and adult cardiomyocytes were isolated from rat hearts. Some of the adult myocytes were cultured to allow for cell dedifferentiation, a phenomenon thought to mimic cell changes that occur in stressed myocardium, with myocytes regressing to a fetal pattern of metabolism and stellate neonatal shape.Using fluorescence deconvolution microscopy, cells were probed with fluorescent markers and scanned for a number of proteins associated with ion control, calcium movements and cardiac function. Image analysis of deconvoluted image stacks and sequential real-time image recordings of calcium transients of cells were made.All three myocyte groups were predominantly comprised of binucleate cells. Clustering of proteins to a single nucleus was a common observation, suggesting that one nucleus is active in protein synthesis pathways, while the other nucleus assumes a 'dormant' or different role and that cardiomyocytes might be mitotically active even in late development, or specific protein syntheses could be targeted and regulated for reintroduction into the cell cycle.Such possibilities would extend cardiac disease associated stem cell research and therapy options, while producing valuable insights into developmental and death pathways of binucleate cardiomyocytes (word count 183).

Cardiomyocyte PDGFR-beta signaling is an essential component of the mouse cardiac response to load-induced stress.

PDGFR is an important target for novel anticancer therapeutics because it is overexpressed in a wide variety of malignancies. Recently, however, several anticancer drugs that inhibit PDGFR signaling have been associated with clinical heart failure. Understanding this effect of PDGFR inhibitors has been difficult because the role of PDGFR signaling in the heart remains largely unexplored. As described herein, we have found that PDGFR-beta expression and activation increase dramatically in the hearts of mice exposed to load-induced cardiac stress. In mice in which Pdgfrb was knocked out in the heart in development or in adulthood, exposure to load-induced stress resulted in cardiac dysfunction and heart failure. Mechanistically, we showed that cardiomyocyte PDGFR-beta signaling plays a vital role in stress-induced cardiac angiogenesis. Specifically, we demonstrated that cardiomyocyte PDGFR-beta was an essential upstream regulator of the stress-induced paracrine angiogenic capacity (the angiogenic potential) of cardiomyocytes. These results demonstrate that cardiomyocyte PDGFR-beta is a regulator of the compensatory cardiac response to pressure overload-induced stress. Furthermore, our findings may provide insights into the mechanism of cardiotoxicity due to anticancer PDGFR inhibitors.

Regulation of Protein Degradation in the Heart by AMP-Activated Protein Kinase

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of protein degradation, and AMPK activity are increased during nutrient deprivation. Pharmacologic and genetic activation of AMPK is sufficient for the induction of MAFbx/Atrogin-1 and MuRF1 in cardiomyocytes and in the heart in vivo. Comprehensive experiments demonstrate that the molecular mechanism by which AMPK regulates MuRF1 expression is through the transcription factor myocyte enhancer factor 2 (MEF2), which is involved in stress response and cardiomyocyte remodeling. MuRF1 is required for AMPK-mediated protein degradation through the UPS in cardiomyocytes. Consequently, the absence of MuRF1 during chronic fasting preserves cardiac function, possibly by limiting degradation of critical metabolic enzymes. Furthermore, during cardiac hypertrophy, chronic activation of AMPK also leads to cardiac dysfunction, possibly through enhanced protein degradation and metabolic dysregulation. Collectively, my findings demonstrate that AMPK regulates expression of ubiquitin ligases which are required for UPS-mediated protein degradation in the heart. Based on these results, I propose that specific metabolic signals may serve as modulators of intracellular protein degradation in the heart.

Regulation of Protein Degradation in the Heart by AMP-Activated Protein Kinase

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of protein degradation, and AMPK activity are increased during nutrient deprivation. Pharmacologic and genetic activation of AMPK is sufficient for the induction of MAFbx/Atrogin-1 and MuRF1 in cardiomyocytes and in the heart in vivo. Comprehensive experiments demonstrate that the molecular mechanism by which AMPK regulates MuRF1 expression is through the transcription factor myocyte enhancer factor 2 (MEF2), which is involved in stress response and cardiomyocyte remodeling. MuRF1 is required for AMPK-mediated protein degradation through the UPS in cardiomyocytes. Consequently, the absence of MuRF1 during chronic fasting preserves cardiac function, possibly by limiting degradation of critical metabolic enzymes. Furthermore, during cardiac hypertrophy, chronic activation of AMPK also leads to cardiac dysfunction, possibly through enhanced protein degradation and metabolic dysregulation. Collectively, my findings demonstrate that AMPK regulates expression of ubiquitin ligases which are required for UPS-mediated protein degradation in the heart. Based on these results, I propose that specific metabolic signals may serve as modulators of intracellular protein degradation in the heart.

Mechanism of molecular regulation of nuclear -encoded mitochondrial gene expression by electrical stimulation in the cultured neonatal rat cardiac myocytes

To answer the question whether increased energy demand resulting from myocyte hypertrophy and enhanced $\beta$-myosin heavy chain mRNA, contractile protein synthesis and assembly leads to mitochondrial proliferation and differentiation, we set up an electrical stimulation model of cultured neonatal rat cardiac myocytes. We describe, as a result of increased contractile activity, increased mitochondrial profiles, cytochrome oxidase mRNA, and activity, as well as a switch in mitochondrial carnitine palmitoyltransferase-I (CPT-I) from the liver to muscle isoform. We investigate physiological pathways that lead to accumulation of gene transcripts for nuclear encoded mitochondrial proteins in the heart. Cardiomyocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation (c-fos, c-jun, junB, nuclear respiratory factor 1 (Nrf-1)), mitochondrial proliferation (cytochrome c (Cyt c), cytochrome oxidase), and mitochondrial differentiation (carnitine palmitonyltransferase I (CPT-I) isoforms) were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25-3 hr) and followed by c-jun (0.5-3 hr), junB (0.5-6 hr), NRF-1 (1-12 hr), Cyt c (12-72 hr), cytochrome c oxidase (12-72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA. Electrical stimulation increased c-fos, $\beta$-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element (CRE), and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the Nrf-1 and CRE sites inhibited the induction by electrical stimulation or by transfection of c-jun into non-paced cardiac myocytes whereas mutation of the Sp-1 site maintained or increased the fold induction. This is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c. Overexpression of c-jun by transfection also activates the Nrf-1 and Cyt c mRNA sequentially. Electrical stimulation of cardiac myocytes activates the c-Jun-N-terminal kinase so that the fold-activation of the cyt c promoter is increased by pacing when either c-jun or c-fos/c-jun are cotransfected. We have identified physical association of Nrf-1 protein with the Nrf-1 enhancer element and of c-Jun with the CRE binding sites on the Cyt c promoter. This is the first demonstration that induction of Nrf-1 and c-Jun by pacing of cardiac myocytes directly mediates Cyt c gene expression and mitochondrial proliferation in response to hypertrophic stimuli in the heart.^ Subsequent to gene activation pathways that lead to mitochondrial proliferation, we observed an isoform switch in CPT-I from the liver to muscle mRNA. We have found that the half-life for the muscle CPT-I is not affected by electrical stimulation, but electrical decrease the T1/2 in the liver CPT-I by greater than 50%. This suggests that the liver CPT-I switch to muscle isoform is due to (1) a decrease in T1/2 of liver CPT-I and (2) activation of muscle CPT-Itranscripts by electrical stimulation. (Abstract shortened by UMI.) ^

Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^