Transgenic Galphaq overexpression induces cardiac contractile failure in mice

Abstract

The critical cell signals that trigger cardiac hypertrophy and regulate the transition to heart failure are not known. To determine the role of Galphaq-mediated signaling pathways in these events, transgenic mice were constructed that overexpressed wild-type Galphaq in the heart using the alpha-myosin heavy chain promoter. Two-fold overexpression of Galphaq showed no detectable effects, whereas 4-fold overexpression resulted in increased heart weight and myocyte size along with marked increases in atrial naturietic factor ( approximately 55-fold), beta-myosin heavy chain ( approximately 8-fold), and alpha-skeletal actin ( approximately 8-fold) expression, and decreased ( approximately 3-fold) beta-adrenergic receptor-stimulated adenylyl cyclase activity. All of these signals have been considered markers of hypertrophy or failure in other experimental systems or human heart failure. Echocardiography and in vivo cardiac hemodynamic studies indeed revealed impaired intrinsic contractility manifested as decreased fractional shortening (19 +/- 2% vs. 41 +/- 3%), dP/dt max, a negative force-frequency response, an altered Starling relationship, and blunted contractile responses to the beta-adrenergic agonist dobutamine. At higher levels of Galphaq overexpression, frank cardiac decompensation occurred in 3 of 6 animals with development of biventricular failure, pulmonary congestion, and death. The element within the pathway that appeared to be critical for these events was activation of protein kinase Cepsilon. Interestingly, mitogen-activated protein kinase, which is postulated by some to be important in the hypertrophy program, was not activated. The Galphaq overexpressor exhibits a biochemical and physiologic phenotype resembling both the compensated and decompensated phases of human cardiac hypertrophy and suggests a common mechanism for their pathogenesis.

Figure 1

Overexpression of Gαq in Gαq-25 transgenic mice results in induction of the hypertrophy gene program. (A) Northern analysis demonstrates expression of a 1.5-kb Gαq specific transcript in ventricles from transgenic mice (+) but not in nontransgenic littermates (−). (B) Western analysis of ventricular protein from transgenic (+) and nontransgenic (−) mice demonstrated 4-fold overexpression of Gαq protein in Gαq-25 transgenic mice. (C) (Left) Gαq-25 (+) ventricular RNA dot blot analysis demonstrates increased transcript levels of β-MHC, ANF, and α-skeletal actin (α SK A), but no significant changes in levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phospholamban (PLB), sarcoplasmic reticulum ATPase (SERCA), cardiac actin (card A), and α-MHC. (Right) Quantitative grouped analysis of mRNA expression normalized to GAPDH expression.

Figure 2

Cell signal activation in Gαq-25 mice. (A) (Top) Western analysis of total, (Middle) tyrosine-204 phosphorylated, and (Bottom) threonine-202/tyrosine-204 dual-phosphorylated MAP kinase. (B) MAP kinase activity assayed by in-gel assay. Serum-starved and 10% serum-stimulated HEK293 cells were used as negative (NC) and positive (PC) controls, respectively. By assessment of either phosphorylation or enzyme activity there is no increase in MAP kinase (p42, p44) activity in Gαq-25 compared with control. Other regulated myelin basic protein kinases (p85, p115) are also not activated in Gαq-25. (C) Western analysis of PKCɛ and PKCα in particulate and cytosolic fractions of ventricular homogenates shows preferential localization of PKCɛ, but not PKCα, to cardiomyocyte particulates in Gαq-25. The ratio of PKCɛ in cardiac membranes/cytosol was 1.2 ± 0.1 in nontransgenic (NTG) and 2.3 ± 0.3 in Gαq-25 (n = 4 pairs, P = 0.02).

Figure 3

Hemodynamic characteristics and left ventricular function of Gαq-25 mice. (A) Force–frequency relations in Gαq-25 mice. Control mice (n = 4, ○) exhibit enhanced left ventricular contractility with increasing heart rate to 500 bpm. Wenckebach heart block developed at rates greater than 600 bpm. ∗, P < 0.05 compared with controls. Gαq-25 mice (n = 4, ▪) showed a negative force–frequency relationship with progressive decline in dP/dt at heart rates greater than 400 bpm. (B) Starling relations in control (n = 4, ○) and Gαq-25 (n = 4, ▪) mice paced at 450 bpm. Intravascular volume expansion was accomplished by intravenous infusion of 400–700 μl of 6% albumin. The Starling relationship is essentially flat for nontransgenics, whereas the Gαq overexpressors are on the “descending limb” at left ventricular end diastolic pressure greater than 7 mmHg. ∗, P < 0.05 compared with baseline. (C and D) Hemodynamic response to βAR stimulation (n = 5 pairs). At matched heart rates of 450 bpm, there is no inotropic (C) or lusitropic (D) response to dobutamine infusion in Gαq transgenic mice, consistent with impaired βAR coupling.

Figure 4

Pathologic examination of a Gαq-25/Gαq-40 dual-heterozygote that developed congestive heart failure (Left) and a nontransgenic sibling (Right). (Top) Gross examination revealed massive cardiomegaly of transgenic heart (×1.8). (Middle) Four-chamber section of hearts (×1.8) demonstrates massive enlargement of both ventricles and atria with atrial filling by organized thrombus. (Bottom) Cardiac histology (left ventricular free wall, ×18) shows mild edema and pale, hypertrophied myocytes without significant inflammation in transgenic heart. Results are representative of three dual-heterozygous mice that exhibited spontaneous cardiac decompensation.