Cardiac myocytes are activated by hormonal and mechanical signals and respond in a variety of ways, from altering contractile function to inducing cardio-protection and growth responses. The use of genetic mouse models allows one to examine the role of cardiac-specific and other genes in cardiac function, hypertrophy, cardio-protection, and diseases such as ischemia and heart failure. However, studies at the cellular level have been hampered by a lack of suitable techniques for isolating and culturing calcium-tolerant, adult mouse cardiac myocytes. We have developed a straightforward, reproducible protocol for isolating and culturing large numbers of adult mouse cardiac myocytes. This protocol is based on the traditional approach of retrograde perfusion of collagenase through the coronary arteries to digest the extracellular matrix of the heart and release rod-shaped myocytes. However, we have made modifications that are essential for isolating calcium-tolerant, rod-shaped adult mouse cardiac myocytes and maintaining them in culture. This protocol yields freshly isolated adult mouse myocytes that are suitable for biochemical assays and for measuring contractile function and calcium transients, and cultured myocytes that are suitable for most biochemical and signaling assays, as well as gene transduction using adenovirus.

Background-In ␣1-AR knockout (␣1ABKO) mice that lacked cardiac myocyte ␣1-adrenergic receptor (␣1-AR) binding, aortic constriction induced apoptosis, dilated cardiomyopathy, and death. However, it was unclear whether these effects were attributable to a lack of cardiac myocyte ␣1-ARs and whether the ␣1A, ␣1B, or both subtypes mediated protection. Therefore, we investigated ␣1A and ␣1B subtype-specific survival signaling in cultured cardiac myocytes to test for a direct protective effect of ␣1-ARs in cardiac myocytes. Methods and Results-We cultured ␣1ABKO myocytes and reconstituted ␣1-AR signaling with adenoviruses expressing ␣1-GFP fusion proteins. Myocyte death was induced by norepinephrine, doxorubicin, or H 2 O 2 and was measured by annexin V/propidium iodide staining. In ␣1ABKO myocytes, all 3 stimuli significantly increased apoptosis and necrosis. Reconstitution of the ␣1A subtype, but not the ␣1B, rescued ␣1ABKO myocytes from cell death induced by each stimulus. To address the mechanism, we examined ␣1-AR activation of extracellular signal-regulated kinase (ERK). In ␣1ABKO hearts, aortic constriction failed to activate ERK, and in ␣1ABKO myocytes, expression of a constitutively active MEK1 rescued ␣1ABKO myocytes from norepinephrine-induced death. In addition, only the ␣1A-AR activated ERK in ␣1ABKO myocytes, and expression of a dominant-negative MEK1 completely blocked ␣1A survival signaling in ␣1ABKO myocytes. Conclusions-Our results demonstrate a direct protective effect of the ␣1A subtype in cardiac myocytes and define an ␣1A-ERK signaling pathway that is required for myocyte survival. Absence of the ␣1A-ERK pathway can explain the failure to activate ERK after aortic constriction in ␣1ABKO mice and can contribute to the development of apoptosis, dilated cardiomyopathy, and death.

No abstract

Abstract-We previously identified an ␣1-AR-ERK (␣1A-adrenergic receptor-extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of ␣1-AR subtypes (␣1A and ␣1B) and how their localization influences ␣1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent ␣1-AR antagonist, we localized endogenous ␣1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify ␣1 subtype localization, we reconstituted ␣1 signaling in cultured ␣1A-and ␣1B-AR double knockout cardiac myocytes using ␣1-AR-green fluorescent protein (GFP) fusion proteins. Similar to endogenous ␣1-ARs and ␣1A-and ␣1B-GFP colocalized with LAP2 at the nuclear membrane. ␣1-AR nuclear localization was confirmed in vivo using ␣1-AR-GFP transgenic mice. The ␣1-signaling partners G␣q and phospholipase C␤1 also colocalized with ␣1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that ␣1-mediated activation of ERK was not inhibited by a membrane impermeant ␣1-blocker, suggesting ␣1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe ␣1-AR localization to caveolae, but we found that ␣1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear ␣1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes. (Circ Res. 2008;103:992-1000.)Key Words: ␣1-adrenergic receptors Ⅲ cardiac myocytes Ⅲ ERK C ardiovascular disease is the leading killer in the United States, accounting for 1.4 million deaths a year. Five million Americans experience heart failure, leading to 970 000 hospitalizations annually, a number that has tripled in the last 25 years. 1 In heart failure, increased activation of the sympathetic nervous system is correlated with pathophysiologic remodeling of the heart, 2 which has led to the therapeutic use of ␤-adrenergic receptor (AR) antagonists in heart failure. However, the general conclusion that inhibition of catecholamine activation of ARs is beneficial in heart failure is disputed by clinical trials with ␣1-AR antagonists. The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) demonstrated that the ␣1-antagonist doxazosin increased the risk of heart failure by 80% and stroke by 26% leading to termination of the trial. 3,4 Similar detrimental effects were seen in the VasodilatorHeart Failure Trials (V-HeFT), in which prazosin was compared with other vasodilators for the prevention of death in heart failure. 5 All 3 ␣1-AR subtypes (A, B, and D) are expressed in the heart 6 -9 ; however, cardiac myocytes only express the ␣1A and ␣1B. 9 Using ␣1A-and ␣1B-AR double knockout mice (␣1ABKO), we demonstrated previously that ␣1-ARs are required for postnatal hypertrophy and adaptation to pathological stress. 9,10 In ␣1ABKO mice, we found that aortic constriction induced dilated cardiomyopathy that le...

Catecholamines and α1-adrenergic receptors (α1-ARs) cause cardiac hypertrophy in cultured myocytes and transgenic mice, but heart size is normal in single KOs of the main α1-AR subtypes, α1A/C and α1B. Here we tested whether α1-ARs are required for developmental cardiac hypertrophy by generating α1A/C and α1B double KO (ABKO) mice, which had no cardiac α1-AR binding. In male ABKO mice, heart growth after weaning was 40% less than in WT, and the smaller heart was due to smaller myocytes. Body and other organ weights were unchanged, indicating a specific effect on the heart. Blood pressure in ABKO mice was the same as in WT, showing that the smaller heart was not due to decreased load. Contractile function was normal by echocardiography in awake mice, but the smaller heart and a slower heart rate reduced cardiac output. α1-AR stimulation did not activate extracellular signal–regulated kinase (Erk) and downstream kinases in ABKO myocytes, and basal Erk activity was lower in the intact ABKO heart. In female ABKO mice, heart size was normal, even after ovariectomy. Male ABKO mice had reduced exercise capacity and increased mortality with pressure overload. Thus, α1-ARs in male mice are required for the physiological hypertrophy of normal postnatal cardiac development and for an adaptive response to cardiac stress

Background— Omega-3 polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) from fish oil ameliorate cardiovascular diseases. However, little is known about the effects of ω-3 polyunsaturated fatty acids on cardiac fibrosis, a major cause of diastolic dysfunction and heart failure. The present study assessed the effects of ω-3 polyunsaturated fatty acids on cardiac fibrosis. Methods and Results— We assessed left ventricular fibrosis and pathology in mice subjected to transverse aortic constriction after the consumption of a fish oil or a control diet. In control mice, 4 weeks of transverse aortic constriction induced significant cardiac dysfunction, cardiac fibrosis, and cardiac fibroblast activation (proliferation and transformation into myofibroblasts). Dietary supplementation with fish oil prevented transverse aortic constriction–induced cardiac dysfunction and cardiac fibrosis and blocked cardiac fibroblast activation. In heart tissue, transverse aortic constriction increased active transforming growth factor-β1 levels and phosphorylation of Smad2. In isolated adult mouse cardiac fibroblasts, transforming growth factor-β1 induced cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid increased cyclic GMP levels and blocked cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid blocked phospho-Smad2/3 nuclear translocation. DT3, a protein kinase G inhibitor, blocked the antifibrotic effects of eicosapentaenoic acid and docosahexaenoic acid. Eicosapentaenoic acid and docosahexaenoic acid increased phosphorylated endothelial nitric oxide synthase and endothelial nitric oxide synthase protein levels and nitric oxide production. Conclusion— Omega-3 fatty acids prevent cardiac fibrosis and cardiac dysfunction by blocking transforming growth factor-β1–induced phospho-Smad2/3 nuclear translocation through activation of the cyclic GMP/protein kinase G pathway in cardiac fibroblasts.

An α 1 -adrenergic receptor (α 1 -AR) antagonist increased heart failure in the Antihypertensive and LipidLowering Treatment to Prevent Heart Attack Trial (ALLHAT), but it is unknown whether this adverse result was due to α 1 -AR inhibition or a nonspecific drug effect. We studied cardiac pressure overload in mice with double KO of the 2 main α 1 -AR subtypes in the heart, α 1A (Adra1a) and α 1B (Adra1b). At 2 weeks after transverse aortic constriction (TAC), KO mouse survival was only 60% of WT, and surviving KO mice had lower ejection fractions and larger end-diastolic volumes than WT mice. Mechanistically, final heart weight and myocyte cross-sectional area were the same after TAC in KO and WT mice. However, KO hearts after TAC had increased interstitial fibrosis, increased apoptosis, and failed induction of the fetal hypertrophic genes. Before TAC, isolated KO myocytes were more susceptible to apoptosis after oxidative and β-AR stimulation, and β-ARs were desensitized. Thus, α 1 -AR deletion worsens dilated cardiomyopathy after pressure overload, by multiple mechanisms, indicating that α 1 -signaling is required for cardiac adaptation. These results suggest that the adverse cardiac effects of α 1 -antagonists in clinical trials are due to loss of α 1 -signaling in myocytes, emphasizing concern about clinical use of α 1 -antagonists, and point to a revised perspective on sympathetic activation in heart failure.

We previously demonstrated that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits myocyte maturation (T. D. O'Connell, D. A. Giacherio, A. K. Jarvis, and R. U. Simpson. Endocrinology 136: 482-488, 1995). To define further the role of 1,25(OH)2D3 in regulating myocardial development, we examined the effects of 1,25(OH)2D3 on proliferation and growth of primary cultures of ventricular myocytes isolated from neonatal rat hearts. When neonatal myocytes were grown in a serum-supplemented medium, cell number approximately doubled, and treating these myocytes with 1,25(OH)2D3 inhibited their proliferation by 56.56% after 4 days. Flow cytometry revealed that 1,25(OH)2D3 reduced the percentage of cells in the S phase of the cell cycle by 31.39% after 4 days. We show for the first time that proliferating cell nuclear antigen protein levels were specifically reduced by 1,25(OH)2D3. Protooncogene c-myc protein levels were also reduced by this hormone. Interestingly, a phorbol ester had a similar effect on myocyte proliferation. Furthermore, 1,25(OH)2D3 increased myocyte protein levels and increased cell size, suggesting that it induces cardiac myocyte hypertrophy. Our findings indicate that 1,25(OH)2D3 and phorbol esters directly regulate myocyte proliferation and induce myocyte hypertrophy. Finally, the data demonstrate that the mechanism by which 1,25(OH)2D3 regulates myocyte proliferation involves blocking entry into the S phase of the cell cycle.