Restoration of contractile function in isolated cardiomyocytes from failing human hearts by gene transfer of SERCA2a

Abstract

Background: Failing human myocardium is characterized by abnormal relaxation, a deficient sarcoplasmic reticulum (SR) Ca(2+) uptake, and a negative frequency response, which have all been related to a deficiency in the SR Ca(2+) ATPase (SERCA2a) pump.

Methods and results: To test the hypothesis that an increase in SERCA2a could improve contractile function in cardiomyocytes, we overexpressed SERCA2a in human ventricular myocytes from 10 patients with end-stage heart failure and examined intracellular Ca(2+) handling and contractile function. Overexpression of SERCA2a resulted in an increase in both protein expression and pump activity and induced a faster contraction velocity (26.7+/-6.7% versus 16.6+/-2.7% shortening per second, P<0.005) and enhanced relaxation velocity (32. 0+/-10.1% versus 15.1+/-2.4%, P<0.005). Diastolic Ca(2+) was decreased in failing cardiomyocytes overexpressing SERCA2a (270+/-26 versus 347+/-30 nmol/L, P<0.005), whereas systolic Ca(2+) was increased (601+/-38 versus 508+/-25 nmol/L, P<0.05). In addition, the frequency response was normalized in cardiomyocytes overexpressing SERCA2a.

Conclusions: These results support the premise that gene-based therapies and targeting of specific pathways in human heart failure may offer a new modality for the treatment of this disease.

Figure 1

a, After isolation, failing human cardiomyocytes were infected with Ad.SERCA2a. Twenty-four hours after infection, a cardiomyocyte is visualized with white light and at 510 nm with single excitation peak at 490 nm of blue light. Coexpression of GFP demonstrates visually that SERCA2a is being expressed in cell. b, Recordings from cardiomyocytes isolated from donor nonfailing heart and from failing heart infected with either Ad.GFP or Ad.SERCA2a, stimulated at 1 Hz at 37°C. Failing cell had a characteristic decrease in contraction and prolonged relaxation along with a prolonged Ca²⁺ transient. Overexpression of SERCA2a in failing cardiomyocyte normalized these parameters. c, Recordings from same cardiomyocytes as in b stimulated at increasing frequencies. Failing cardiomyocyte demonstrated a decrease in contraction amplitude and an increase in diastolic tone and Ca²⁺. Overexpression of SERCA2a restored frequency-dependent increase in contraction amplitude and mitigated increase in diastolic Ca²⁺ and length.

Figure 2

a, Immunoblots of SERCA2a from crude membranes of failing cardiomyocytes infected for 24 hours with Ad.SERCA2a or Ad.GFP. All representative lanes are from failing hearts. Each pair of immunoreactive densities represents paired immunoblots from 1 preparation of failing cardiomyocytes that were infected with either Ad.GFP or Ad.SERCA2a. b, Paired measurements of ATPase activity performed at [Ca²⁺] of 10 μmol/L in membrane preparations from failing cardiomyocytes infected for 24 hours with Ad.SERCA2a or Ad.GFP.