Rationale Induction of the fetal hypertrophic marker gene beta-myosin heavy chain (β-MyHC) is a signature feature of pressure overload hypertrophy in rodents. β-MyHC is assumed present in all or most enlarged myocytes. Objective To quantify the number and size of myocytes expressing endogenous β-MyHC using a flow cytometry approach. Methods and Results Myocytes were isolated from the LV of male C57Bl/6J mice after transverse aortic constriction (TAC), and the fraction of cells expressing endogenous β-MyHC was quantified by flow cytometry on 10,000–20,000 myocytes, using a validated β-MyHC antibody. Side scatter by flow cytometry in the same cells was validated as an index of myocyte size. β-MyHC-positive myocytes were 3±1% of myocytes in control hearts (n=12), increasing to 25±10% at 3d-6w after TAC (n=24, p<0.01). β-MyHC-positive myocytes did not enlarge with TAC, and were smaller at all times than myocytes without β-MyHC (~70% as large, p<0.001). β-MyHC-positive myocytes arose by addition of β-MyHC to α-MyHC, and had more total MyHC after TAC than did the hypertrophied myocytes that had α-MyHC only. Myocytes positive for β-MyHC were found in discrete regions of the LV, in 3 patterns, peri-vascular, in areas with fibrosis, and in apparently normal myocardium. Conclusion β-MyHC protein is induced by pressure overload in a minor sub-population of smaller cardiac myocytes. The hypertrophied myocytes after TAC have α-MyHC only. These data challenge the current paradigm of the fetal hypertrophic gene program, and identify a new sub-population of smaller working ventricular myocytes with more myosin.

Rationale It is unknown if every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), beta-1, beta-2, beta-3, alpha-1A, and alpha-1B. The beta-1 and beta-2 are thought to be the dominant myocyte ARs. Objective Quantify the 5 cardiac ARs in individual ventricular myocytes. Methods and Results We studied ventricular myocytes from wild type mice, mice with alpha-1A and alpha-1B knockin reporters, and beta-1 and beta-2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of ERK and phospholamban), and contraction. We found that the beta-1 and alpha-1B were present in all myocytes. The alpha-1A was present in 60%, with high levels in 20%. The beta-2 and beta-3 were detected in only about 5% of myocytes, mostly in different cells. In intact heart, 30% of total beta-ARs were beta-2 and 20% were beta-3, both mainly in nonmyocytes. Conclusion The dominant ventricular myocyte ARs present in all cells are the beta-1 and alpha-1B. The beta-2 and beta-3 are mostly absent in myocytes but are abundant in nonmyocytes. The alpha-1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with beta-1 and alpha-1B only; 60% that also have the alpha-1A; and 5% each that also have the beta-2 or beta-3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms.