Protein kinase (PK)Calpha, PKCbeta, and PKCgamma comprise the conventional PKC isoform subfamily, which is thought to regulate cardiac disease responsiveness. Indeed, mice lacking the gene for PKCalpha show enhanced cardiac contractility and reduced susceptibility to heart failure. Recent data also suggest that inhibition of conventional PKC isoforms with Ro-32-0432 or Ro-31-8220 enhances heart function and antagonizes failure, although the isoform responsible for these effects is unknown. Here, we investigated mice lacking PKCalpha, PKCbeta, and PKCgamma for effects on cardiac contractility and heart failure susceptibility. PKCalpha(-/-) mice, but not PKCbetagamma(-/-) mice, showed increased cardiac contractility, myocyte cellular contractility, Ca(2+) transients, and sarcoplasmic reticulum Ca(2+) load. PKCalpha(-/-) mice were less susceptible to heart failure following long-term pressure-overload stimulation or 4 weeks after myocardial infarction injury, whereas PKCbetagamma(-/-) mice showed more severe failure. Infusion of ruboxistaurin (LY333531), an orally available PKCalpha/beta/gamma inhibitor, increased cardiac contractility in wild-type and PKCbetagamma(-/-) mice, but not in PKCalpha(-/-) mice. More importantly, ruboxistaurin prevented death in wild-type mice throughout 10 weeks of pressure-overload stimulation, reduced ventricular dilation, enhanced ventricular performance, reduced fibrosis, and reduced pulmonary edema comparable to or better than metoprolol treatment. Ruboxistaurin was also administered to PKCbetagamma(-/-) mice subjected to pressure overload, resulting in less death and heart failure, implicating PKCalpha as the primary target of this drug in mitigating heart disease. As an aside, PKCalphabetagamma triple-null mice showed no defect in cardiac hypertrophy following pressure-overload stimulation. In conclusion, PKCalpha functions distinctly from PKCbeta and PKCgamma in regulating cardiac contractility and heart failure, and broad-acting PKC inhibitors such as ruboxistaurin could represent a novel therapeutic approach in treating human heart failure.